Acid-cleavable linkers exhibiting altered rates of acid hydrolysis

ABSTRACT

An acid-cleavable peptide linker comprising aspartic acid and proline residues is disclosed. The acid-cleavable peptide linker provides an altered sensitivity to acid-hydrolytic release of peptides of interest from fusion peptides of the formula PEP1-L-PEP2. The inventive linker, L, is described in various embodiments, each of which provides substantially more rapid acid-release of peptides of interest than does a single aspartic acid-proline pair. In an additional aspect, a method of increasing the stability of an acid cleavable linkage to acid hydrolysis is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 14/077,342, filed Nov. 12, 2013, which is a divisional of U.S.patent application Ser. No. 14/077,342, filed Sep. 7, 2011, whichgranted U.S. Pat. No. 8,609,621, which claims benefit of expired U.S.Provisional Patent Application No. 61/413,501, filed Nov. 15, 2010, eachof which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates to the field of protein expression andpurification from microbial cells. More specifically, peptide linkershaving an altered sensitivity to acid hydrolysis are provided as well asmethods of their use.

BACKGROUND OF THE INVENTION

The efficient production of bioactive proteins and peptides has become ahallmark of the biomedical and industrial biochemical industry.Bioactive peptides and proteins are used as curative agents in a varietyof diseases such as diabetes (insulin), viral infections and leukemia(interferon), diseases of the immune system (interleukins), and redblood cell deficiencies (erythropoietin) to name a few. Additionally,large quantities of proteins and peptides are needed for variousindustrial applications including, for example, the pulp and paper andpulp industries, textiles, food industries, personal care and cosmeticsindustries, sugar refining, wastewater treatment, production ofalcoholic beverages and as catalysts for the generation of newpharmaceuticals.

With the advent of the discovery and implementation of combinatorialpeptide screening technologies such as bacterial display, yeast display,phage display, ribosome display, and mRNA display technology newapplications for peptides having strong affinity for a target surfacehave been developed. In particular, peptides are being looked to aslinkers in biomedical fields for the attachment of diagnostic andpharmaceutical agents to surfaces (see Grinstaff et al, U.S. PatentApplication Publication No. 2003-0185870 and Linter in U.S. Pat. No.6,620,419), as well as in the personal care industry for the attachmentof benefit agents to body surfaces such as hair and skin (see commonlyowned U.S. Pat. No. 7,220,405, and Janssen et al. U.S. Pat. No.7,129,326), and in the printing industry for the attachment of pigmentsto print media (see commonly owned U.S. Patent Application PublicationNo. 2005-0054752).

In some cases commercially useful proteins and peptides may besynthetically generated or isolated from natural sources. However, thesemethods are often expensive, time consuming and characterized by limitedproduction capacity. The preferred method of protein and peptideproduction is through the fermentation of recombinantly constructedorganisms, engineered to over-express the protein or peptide ofinterest. Although preferable to synthesis or isolation, recombinantexpression of peptides has a number of obstacles to be overcome in orderto be a cost-effective means of production. For example, peptides (andin particular short peptides) produced in a cellular environment aresusceptible to degradation from the action of native cellular proteases.Additionally, purification can be difficult, resulting in poor yieldsdepending on the nature of the protein or peptide of interest.

One means to mitigate the above difficulties is the use of geneticchimera for protein and peptide expression. A chimeric protein or“fusion protein” is a polypeptide comprising at least one portion of adesired protein product fused to at least one portion comprising apeptide tag. The peptide tag may be used to assist protein folding,assist in purification, alter polypeptide solubility, protect theprotein from the action of degradative enzymes, and/or assist theprotein in various transport and targeting processes.

In many cases it is useful to express a protein or peptide in insolubleform, particularly when the peptide of interest is rather short,normally soluble, and/or subject to proteolytic degradation within thehost cell. Production of the peptide in insoluble form both facilitatessimple recovery and protects the peptide from undesirable proteolyticdegradation. One means to produce the peptide in insoluble form is torecombinantly produce the peptide as part of an insoluble fusion proteinby including in the fusion construct, at least one peptide tag (i.e., aninclusion body tag) that induces inclusion body formation. Typically,the fusion protein is designed to include at least one cleavable peptidelinker so that the peptide of interest can be subsequently recoveredfrom the fusion protein. The fusion protein may be designed to include aplurality of solubility tags, cleavable peptide linkers, and regionsencoding the peptide of interest.

Fusion proteins comprising a peptide tag that facilitate the expressionof insoluble proteins are well known in the art. Typically, the tagportion of the chimeric or fusion protein is large, increasing thelikelihood that the fusion protein will be insoluble. Examples of largepeptides that are typically used include, but are not limited tochloramphenicol acetyltransferase (Dykes et al., Eur. J. Biochem.,174:411 (1988), β-galactosidase (Schellenberger et al., Int. J. PeptideProtein Res., 41:326 (1993); Shen et al., Proc. Nat. Acad. Sci. USA281:4627 (1984); and Kempe et al., Gene, 39:239 (1985)),glutathione-S-transferase (Ray et al., Bio/Technology, 11:64 (1993) andHancock et al. (WO94/04688)), the N-terminus of L-ribulokinase (U.S.Pat. No. 5,206,154 and Lai et al., Antimicrob. Agents & Chemo., 37:1614(1993), bacteriophage T4 gp55 protein (Gramm et al., Bio/Technology,12:1017 (1994), bacterial ketosteroid isomerase protein (Kuliopulos etal., J. Am. Chem. Soc. 116:4599 (1994), ubiquitin (Pilon et al.,Biotechnol. Prog., 13:374-79 (1997), bovine prochymosin (Naught et al.,Biotechnol. Bioengineer. 57:55-61 (1998), andbactericidal/permeability-increasing protein (“BPI”; Better, M. D. andGavit, PD., U.S. Pat. No. 6,242,219). The art is replete with specificexamples of this technology, see for example U.S. Pat. No. 6,613,548,describing fusion protein of proteinaceous tag and a soluble protein andsubsequent purification from cell lysate; U.S. Pat. No. 6,037,145,teaching a tag that protects the expressed chimeric protein from aspecific protease; U.S. Pat. No. 5,648,244, teaching the synthesis of afusion protein having a tag and a cleavable linker for facilepurification of the desired protein; and U.S. Pat. No. 5,215,896; U.S.Pat. No. 5,302,526; and U.S. Pat. No. 5,330,902; and U.S. PatentApplication Publication No. 2005-221444, describing fusion tagscontaining amino acid compositions specifically designed to increaseinsolubility of the chimeric protein or peptide.

Shorter solubility tags have been developed from the Zea mays zeinprotein (co-owned U.S. Pat. No. 7,732,569) the Daucus carota cystatin(co-owned U.S. Pat. No. 7,662,913), and an amyloid-like hypotheticalprotein from Caenorhabditis elegans (co-owned U.S. Pat. No. 7,427,656;each hereby incorporated by reference in their entirety.) The use ofshort inclusion body tags increases the yield of the target peptideproduced within the recombinant host cell.

Aspartic acid-proline linkages can be cleaved using acid treatment.However, the conditions typically used include at least one strong acid,such as HCl or H₂SO₄, and may require subsequent neutralization withbase and may increase the cost of peptide recovery due the amount ofsalt produced. Further, acid hydrolysis conditions for the intendedaspartic acid-proline pair may be accompanied by undesirable hydrolysisat other sites where aspartic acid residues occur or may lead to thedeamidation of glutamine or asparagine.

One problem to be solved is to provide peptide linkers that are moresensitive to acid hydrolysis when compared to a single asparticacid-proline linkage. Increased sensitivity may permit the use of weakeracids, reduce the amount of base that may be needed for neutralization,and may help to protect the peptide of interest from unwanted hydrolysisat other locations within the peptide of interest.

Situations may occur where a peptide or protein of interest contains oneor more acid labile aspartic acid-proline linkages where acid hydrolysisis not desired. As such, another problem to be solved is to provide amethod to increase the stability of aspartic acid-proline linkages toacid treatment in peptides or proteins wherein acid hydrolysis isundesirable.

SUMMARY OF THE INVENTION

The stated problem has been solved though the discovery of peptidelinkers characterized by increased sensitivity to acid hydrolysis whencompared to a single aspartic acid-proline (i.e., DP) linkage.

In one embodiment, peptide linkers characterized by greater sensitivityto acid treatment are provided, wherein the peptide linkers comprise anamino acid sequence selected from the group consisting of:

(SEQ ID NO: 1) A. DPDP (SEQ ID NO: 2) B. DPDPDP (SEQ ID NO: 3)C. DPDPDPDP (SEQ ID NO: 4) D. DPDPDPP (SEQ ID NO: 5) E. DPDPPDPP(SEQ ID NO: 6) F. DPDPPDP, and (SEQ ID NO: 7) G. DPPDPPDP,

wherein D is aspartic acid and P is proline.

In a further embodiment, the invention disclosed herein encompasses afusion peptide according to the structure PEP1-L-PEP2, wherein,

-   -   a) PEP1 and PEP2 are independently functional peptides wherein        at least one is a peptide of interest (“POI”); and    -   b) L is an acid-cleavable linker comprising a peptide selected        from the group consisting of:

(SEQ ID NO: 1) A. DPDP, (SEQ ID NO: 2) B. DPDPDP, (SEQ ID NO: 3)C. DPDPDPDP, (SEQ ID NO: 4) D. DPDPDPP, (SEQ ID NO: 5) E. DPDPPDPP,(SEQ ID NO: 6) F. DPDPPDP, and (SEQ ID NO: 7) G. DPPDPPDP,

wherein D is aspartic acid and P is proline.

The linker, L, provides for increased efficiency of acid release of apeptide from a fusion peptide comprising L. This increase may be definedas the enhancement in acid hydrolysis rate of an intact fusion peptidehaving a linker of the formula according to the invention when comparedto the acid hydrolysis rate of the fusion peptide, PEP1-L-PEP2, having asingle DP pair as the linker, L.

In this embodiment, the peptides PEP1 and PEP2 may each be a peptide ofinterest (i.e., “POI”). Alternatively, one of PEP1 or PEP2 may be aninclusion body tag (i.e., “IBT”) whereas the remaining peptide may be aPOI. In the context of the present invention an IBT is a peptide orpolypeptide that directs newly synthesized fusion peptide molecules toprecipitate or accumulate in insoluble inclusion bodies that can form inrecombinant cells expressing heterologous, i.e., foreign,polynucleotides encoding peptides, polypeptides, fusion peptides and thelike.

The POI of the fusion peptide may be virtually any peptide orpolypeptide. The POI may be one of many targeting peptides that areidentifiable by known biopanning methods after their expression in arecombinant bacteriophage. Such targeting peptides have high affinityfor various targets of interest, including but not limited to skin,hair, nails, print media, woven or nonwoven fabric, polymers, toothenamel, tooth pellicle, and clay and the like. POIs may also includeantimicrobial peptides, pigment-binding peptides, and cellulose-bindingpeptides.

Such POIs may have functional applications such as diagnostic markers,pharmaceuticals, stimulators or inhibitors of enzymatic orreceptor-mediated processes, and the like. Thus, the fusion peptidecomprising the linker L can be employed in an isolation and purificationscheme for any POI or polypeptide that can be cleaved from the fusionpeptide as a result of being contacted with a sufficiently acidicenvironment.

In one embodiment, the invention encompasses a method of isolating apeptide of interest (“POI”) from a recombinant cell expressing aheterologous fusion peptide comprising the linker L. The recombinantcell may be any prokaryotic or eukaryotic cell type, including any typeof recombinant microbial cell. Preferred recombinant microbial cellsinclude recombinant yeast cells and recombinant bacterial cells. Anadditional embodiment contemplates taking advantage of the fact thatheterologous peptide expression in recombinant cells is oftenaccompanied by the newly synthesized heterologous peptides accumulatingin insoluble inclusion bodies within the nucleus or the cytoplasm of thecell. When such insoluble fusion peptides having a POI also comprise theacid-cleavable linker L, it is contemplated herein that acid hydrolysisof L will liberate the POI from the inclusion body, preferablyconverting it to a more soluble form. The released soluble form of thePOI is then more easily separable from remaining inclusion bodies andinsoluble remnants thereof.

However, the fusion peptide is equally suitable to embodiments whereinthe chemistry of PEP1 and PEP2 result in the synthesis of a solublefusion peptide that remains soluble in the cytoplasm and does notprecipitate or form inclusion bodies. In such cases the acid cleavablelinker provides a simple method to separate a soluble POI from thesoluble fusion peptide or cleaved fragment thereof.

Thus, an additional embodiment of the invention comprises a method ofpreparing at least one peptide of interest (“POI”) from a fusion peptidecomprising the at least one POI, comprising:

a) providing a recombinant cell synthesizing a fusion peptide having thestructure

PEP1-L-PEP2

wherein,

-   -   i) PEP1 and PEP2 are independently functional peptides wherein        at least one is a peptide of interest (“POI”); and    -   ii) L is an acid-cleavable linker comprising a peptide selected        from the group consisting of:

(SEQ ID NO: 1) A. DPDP, (SEQ ID NO: 2) B. DPDPDP, (SEQ ID NO: 3)C. DPDPDPDP, (SEQ ID NO: 4) D. DPDPDPP, (SEQ ID NO: 5) E. DPDPPDPP,(SEQ ID NO: 6) F. DPDPPDP, and (SEQ ID NO: 7) G. DPPDPPDP,

wherein D is aspartic acid and P is proline; and

b) contacting the fusion peptide with a solution of sufficiently acidicpH so that linker L is cleaved, and

c) isolating the at least one POI.

In an even further embodiment, the invention encompasses a recombinantcell, preferably a microbial cell, and more preferably a bacterial cellthat expresses such a fusion peptide. In this context an especiallydesirable bacterial cell is E. coli.

Situations may exist where there is a need to increase the stability ofa peptide or protein comprising at least one aspartic acid-prolinelinkage to an acid treatment. As such, a method to increase thestability of an acid cleavable linkage to acid hydrolysis is alsoprovided comprising:

-   -   a) providing a peptide or protein of interest comprising at        least one acid cleavable linkage having the following structure:        -   XDP;        -   wherein D is aspartic acid and P is proline and X is any            amino acid other than tryptophan or phenylalanine; and    -   b) altering said at least one acid cleavable linkage by        substituting X with tryptophan or phenylalanine; whereby the        stability of the acid cleavable linkage to acid hydrolysis is        increased by the substitution.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the relative rates of acid cleavage of an intact fusionpeptide having a DP cleavage site (“DP1”), a DPDP cleavage site (“DP2”;SEQ ID NO: 1), a DPDPDP cleavage site (“DP3”; SEQ ID NO: 2) or aDPDPDPDP cleavage site (“DP4”; SEQ ID NO: 3).

FIG. 2 shows a plot of the halftime, t_(1/2), of acid hydrolysisperformed at about 70° C. as a function of the number of DP pairs in theacid cleavable linker.

FIG. 3 demonstrates the effect on acid hydrolysis rates of an intactfusion peptide having an amino acid substitution in the positionimmediately to the amino terminal side of the aspartic acid residue of asingle DP linker; i.e., an aspartic acid-proline pair.

FIG. 4 demonstrates the effect on acid hydrolysis rates of an intactfusion peptide having an amino acid substitution in the positionimmediately to the carboxy terminal side of the proline residue of asingle DP linker; i.e., an aspartic acid-proline pair.

FIG. 5 demonstrates the effect on rates of acid hydrolysis of an intactfusion peptide of having additional proline residues included in theacid cleavable linker “DP3”; DP3 (SEQ ID NO: 2), PP1 (SEQ ID NO: 4), PP2(SEQ ID NO: 5) and PP3 (SEQ ID NO: 6).

FIG. 6 demonstrates the effect on rates of acid hydrolysis of an intactfusion peptide of placing additional proline residues within the acidcleavable linker “DP3”; DP3 (SEQ ID NO: 2), PP2 (SEQ ID NO: 5) and PP4(SEQ ID NO: 7). See Table 2 for each corresponding linker sequence.

FIG. 7 demonstrates the effect of different acidic conditions on therate of acid hydrolysis of an intact fusion peptide comprising the acidcleavable linker DP1, DP3, PP2 or PP3 (SEQ ID NO: 5) at approximately70° C. See Table 2 for each corresponding linker sequence.

FIG. 8 demonstrates the effect of different acidic conditions on therate of acid hydrolysis of an intact fusion peptide having either theacid cleavable linker DP3 (SEQ ID NO: 2) or PP2 (SEQ ID NO: 5) atreduced temperature of 50° C. See Table 2 for each corresponding linkersequence.

FIG. 9 demonstrates the effect of different acidic conditions on theextent of acid hydrolysis of an intact fusion peptide KSI(C4E).DP.HC353having the acid cleavable linker DP. Maximum cleavage is obtained after4 h at pH 2 and 80° C. Arrows indicate the full length fusion (F), HC353(H) and KSI(C4E) (K).

FIG. 10 demonstrates the effect of different acidic conditions on theextent of acid hydrolysis of an intact fusion peptideKSI(C4E).DPDPPDPP.HC353 having the acid cleavable linker DPDPPDPP.Maximum cleavage is obtained after only 1 h at pH 2 and 80° C., at 60°C. for 4 h at pH 2 or at pH 4 for 4 hr and at 80° C. Arrows indicate thefull length fusion (F), HC353 (H) and KSI(C4E) (K).

FIG. 11 demonstrates the effect of different acidic conditions on theextent of acid hydrolysis of an intact fusion peptideKSI(C4E).DPPDPPDP.HC353 having the acid cleavable linker DPPDPPDP.Maximum cleavage is obtained after only 90 min 4 h at pH 2 and 80° C.,at 70° C. for 4 h at pH 2 or at pH 3 for 4 h and at 80° C. Arrowsindicate the full length fusion (F), HC353 (H) and KSI(C4E) (K).

FIG. 12 demonstrates the effect of different acidic conditions on theextent of acid hydrolysis of an intact fusion peptideKSI(C4E).DPDPDP.HC353 having the acid cleavable linker DPDPDP. Maximumcleavage is obtained after only 120 min 4 h at pH 2 and 80° C., at 70°C. for 4 h at pH 2 or at pH 3 for 4 h and at 80° C. Arrows indicate thefull length fusion (F), HC353 (H) and KSI(C4E) (K).

BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

The following sequences comply with 37 C.F.R. 1.821-1.825 (“Requirementsfor Patent Applications Containing Nucleotide Sequences and/or AminoAcid Sequence Disclosures—the Sequence Rules”) and are consistent withWorld Intellectual Property Organization (WIPO) Standard ST.25 (2009)and the sequence listing requirements of the EPC and PCT (Rules 5.2 and49.5(a-bis), and Section 208 and Annex C of the AdministrativeInstructions). The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NOs: 1-7 are the amino acid sequences of various embodiments ofthe present acid-cleavable linkers.

SEQ ID NOs: 8-25 are the polynucleotide sequences of the primers,oligonucleotides and plasmids used in preparing the polynucleotidesencoding recombinant fusion peptides INK101, INK101DP, INK101DP2,INK101DP3, and INK101DP4.

SEQ ID NO: 26 is the amino acid sequence of the core acid-cleavablepeptide, INK101DP.

SEQ ID NOs: 27-102 are primer sequences for performing mutagenesis ofthe immediately amino- and carboxy-terminal neighboring amino acids ofthe DP pair in INK101DP.

SEQ ID NOs: 103-110 are mutagenesis primer sequences that direct theinsertion of additional proline residues into the acid-cleavable linkerof INK101DP3.

SEQ ID NOs: 111-235 are amino acid sequences of various target-specificbinding peptides as provided in Table 1 below:

TABLE 1 SEQ ID NOs: Target Specificity 111-121 Hair 122-132 Skin 133-134Finger/toe nail 135-143 Tooth (pellicle) 144-154 Tooth (enamel) 155-161Antimicrobial 162-172 Clay 173-185 Calcium carbonate 186-192Polypropylene 193-201 Polytetrafluoroethylene 202-208 Polyethylene209-214 Nylon 215-217 Polystyrene 218-221 Cellulose acetate 222-225Carbon black 226-230 Cromophtal yellow 231-235 Sunfast magenta

SEQ ID NOs: 236-249 are the amino acid sequences of peptides thatfunction as inclusion body tags (IBTs).

SEQ ID NO: 250 is plasmid PLX121 that provides for expression of theINK101DP peptide.

SEQ ID NO: 251 is the sequence of the polynucleotide encoding theINK101DP peptide.

SEQ ID NO: 252 is the amino acid sequence of solubility tag KSI(C4E).

SEQ ID NO: 253 is the amino acid sequence of the peptide of interestHC353.

SEQ ID NO: 254 is the nucleic acid sequence of plasmid pLD001. SEQ IDNO: 255 is the nucleic acid sequence encoding fusion peptide

KSI(C4E).DP.HC353.

SEQ ID NO: 256 is the amino acid sequence of fusion peptideKSI(C4E).DP.HC353.

SEQ ID NO: 257 is the nucleic acid sequence of primer 353.DP3 UP.

SEQ ID NO: 258 is the nucleic acid sequence of primer 353.DP3 DOWN.

SEQ ID NO: 259 is the nucleic acid sequence encoding fusion peptideKSI(C4E).DPDPDP.HC353.

SEQ ID NO: 260 is the amino acid sequence of fusion peptideKSI(C4E).DPDPDP.HC353.

SEQ ID NO: 261 is the nucleic acid sequence of primer PP2 HC353 UP.

SEQ ID NO: 262 is the nucleic acid sequence of primer PP2 HC353 DOWN.

SEQ ID NO: 263 is the nucleic acid sequence encoding fusion peptideKSI(C4E).DPDPPDPP.HC353.

SEQ ID NO: 264 is the amino acid sequence of fusion peptideKSI(C4E).DPDPPDPP.HC353.

SEQ ID NO: 265 is the nucleic acid sequence of primer 353 PP4 UP.

SEQ ID NO: 266 is the nucleic acid sequence of primer 353 PP4 DOWN.

SEQ ID NO: 267 is the nucleic acid sequence encoding fusion peptideKSI(C4E).DPPDPPDP.HC353.

SEQ ID NO: 268 is the amino acid sequence of fusion peptideKSI(C4E).DPPDPPDP.HC353.

Several of the peptides listed above and the methods by which they wereidentified and prepared have been previously described in detail in U.S.Patent Application Publication Nos. U.S. 2009-0048428 and U.S.2005-0054752, both of which are hereby incorporated by reference.

Persons of ordinary skill in the art will readily appreciate that theforegoing non-limiting listing of distinct classes of peptides isprovided for illustrative purposes only, as examples of the scope ofdistinct sets of targeting peptides that may be incorporated into afusion peptide of the formula PEP1-L-PEP2 wherein L is an acid-cleavablelinker encompassed by the invention disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used herein and should be referred to forinterpretation of the claims and the specification. Unless otherwisenoted, all U.S. patents and U.S. patent applications referenced hereinare incorporated by reference in their entirety.

As used herein, the articles “a”, “an”, and “the” preceding an elementor component of the invention are intended to be nonrestrictiveregarding the number of instances (i.e., occurrences) of the element orcomponent. Therefore “a”, “an”, and “the” should be read to include oneor at least one, and the singular word form of the element or componentalso includes the plural unless the number is obviously meant to besingular.

As used herein, the term “comprising” means the presence of the statedfeatures, integers, steps, or components as referred to in the claims,but that it does not preclude the presence or addition of one or moreother features, integers, steps, components or groups thereof. The term“comprising” is intended to include embodiments encompassed by the terms“consisting essentially of” and “consisting of”. Similarly, the term“consisting essentially of” is intended to include embodimentsencompassed by the term “consisting of”′.

As used herein, the term “about” modifying the quantity of an ingredientor reactant employed refers to variation in the numerical quantity thatcan occur, for example, through typical measuring and liquid handlingprocedures used for making concentrates or use solutions in the realworld; through inadvertent error in these procedures; throughdifferences in the manufacture, source, or purity of the ingredientsemployed to make the compositions or carry out the methods; and thelike. The term “about” also encompasses amounts that differ due todifferent equilibrium conditions for a composition resulting from aparticular initial mixture. Whether or not modified by the term “about”,the claims include equivalents to the quantities.

Where present, all ranges are inclusive and combinable. For example,when a range of “1 to 5” is recited, the recited range should beconstrued as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”,“1-3 & 5”, and the like.

As used herein, the term “isolated nucleic acid molecule” is a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid molecule in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

As used herein, the term “pigment” refers to an insoluble, organic orinorganic colorant.

As used herein, the term “hair” as used herein refers to human hair,eyebrows, and eyelashes.

As used herein, the term “skin” as used herein refers to human skin, orsubstitutes for human skin, such as pig skin, VITRO-SKIN® and EPIDERM™.Skin, as used herein, will refer to a body surface generally comprisinga layer of epithelial cells and may additionally comprise a layer ofendothelial cells.

As used herein, the term “nails” as used herein refers to humanfingernails and toenails.

As used herein, “PBP” means polymer-binding peptide. As used herein, theterm “polymer-binding peptide” refers to peptide sequences that bindwith high affinity to a specific polymer (U.S. Pat. No. 7,427,656).Examples include peptides that bind to polyethylene (SEQ ID NO:202-208),polypropylene (SEQ ID NOs: 186-192), polystyrene (SEQ ID NOs: 215-217),Nylon (SEQ ID NOs: 209-214), and poly(tetrafluoroethylene) (SEQ ID NOs:193-201).

As used herein, “HBP” means hair-binding peptide. As used herein, theterm “hair-binding peptide” refers to peptide sequences that bind withhigh affinity to hair. The hair-binding peptide may be comprised of asingle hair-binding domain or multiple binding domains wherein at leastone of the binding-domains binds to hair (i.e. multi-block peptides).Examples of hair binding peptides have been reported (U.S. patentapplication Ser. No. 11/074,473 to Huang et al.; WO 0179479; U.S. PatentApplication Publication No. 2002/0098524 to Murray et al.; Janssen etal., U.S. Patent Application Publication No. 2003/0152976 to Janssen etal.; WO 2004048399; U.S. application Ser. No. 11/512,910, and U.S.patent application Ser. No. 11/696,380). Examples of hair-bindingpeptides are provided as SEQ ID NOs: 111-121.

As used herein, “SBP” means skin-binding peptide. As used herein, theterm “skin-binding peptide” refers to peptide sequences that bind withhigh affinity to skin. Examples of skin binding peptides have also beenreported (U.S. patent application Ser. No. 11/069,858 toBuseman-Williams; Rothe et. al., WO 2004/000257; and U.S. patentapplication Ser. No. 11/696,380). Skin as used herein as a body surfacewill generally comprise a layer of epithelial cells and may additionallycomprise a layer of endothelial cells. Examples of skin-binding peptidesare provided as SEQ ID NOs: 122-132.

As used herein, “NBP” means nail-binding peptide. As used herein, theterm “nail-binding peptide” refers to peptide sequences that bind withhigh affinity to nail. Examples of nail binding peptides have beenreported (U.S. patent application Ser. No. 11/696,380). Examples ofnail-binding peptides are provided as SEQ ID NOs: 133-134.

As used herein, an “antimicrobial peptide” is a peptide having theability to kill microbial cell populations (U.S. Pat. No. 7,427,656).Examples of antimicrobial peptides are provided as SEQ ID NOs: 155-161.

As used herein, “cellulose acetate-binding peptide” refers to a peptidethat binds with high affinity to cellulose acetate. Examples ofcellulose acetate-binding peptides are provided as SEQ ID NOs: 218-221.

As used herein, “clay-binding peptide” refers to a peptide that bindswith high affinity to clay (U.S. patent application Ser. No.11/696,380). Examples of clay-binding peptides are provided as SEQ IDNOs: 162-172.

As used herein, “calcium carbonate-binding peptide” refers to a peptidethat binds with high affinity to calcium carbonate. Examples of calciumcarbonate-binding peptides are provided as SEQ ID NOs: 173-185.

As used herein, “tooth-pellicle-binding peptide” refers to a peptidethat binds with high affinity to the proteinaceous tooth pellicle layerthat lies external to the enamel. Examples of tooth pellicle-bindingpeptides are provided as SEQ ID NOs: 135-143.

As used herein, “tooth-enamel-binding peptide” refers to a peptide thatbinds with high affinity to the enamel of the tooth. Examples of toothenamel-binding peptides are provided as SEQ ID NOs: 144-154.

As used herein, “pigment-binding peptide” refers to a peptide that bindswith high affinity to pigment particles of various types. Examples ofpigment-binding peptides are peptides that bind to carbon black (SEQ IDNOs: 222-225), Cromophtal yellow (SEQ ID NOs: 226-230) and SunfastMagenta (SEQ ID NOs: 231-235).

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). In a furtherembodiment, the definition of “operably linked” may also be extended todescribe the products of chimeric genes, such as fusion peptides. Assuch, “operably linked” will also refer to the linking of an inclusionbody tag to a peptide of interest to be produced and recovered. Theinclusion body tag is “operably linked” to the peptide of interest ifupon expression the fusion protein is insoluble and accumulates asinclusion bodies in the expressing host cell.

Means to prepare the present peptides are well known in the art (see,for example, Stewart et al., Solid Phase Peptide Synthesis, PierceChemical Co., Rockford, Ill., 1984; Bodanszky, Principles of PeptideSynthesis, Springer-Verlag, New York, 1984; and Pennington et al.,Peptide Synthesis Protocols, Humana Press, Totowa, N.J., 1994). Thevarious components of the fusion peptides (inclusion body tag, peptideof interest, and the cleavable linker/cleavage sequence) describedherein can be combined using carbodiimide coupling agents (see forexample, Hermanson, Greg T., Bioconjugate Techniques, Academic Press,New York (1996)), diacid chlorides, diisocyanates and other difunctionalcoupling reagents that are reactive to terminal amine and/or carboxylicacid groups on the peptides. However, chemical synthesis is oftenlimited to peptides of less than about 50 amino acids length due to costand/or impurities. In a preferred embodiment, the biological moleculesdescribed herein are prepared using standard recombinant DNA andmolecular cloning techniques.

As used herein, the terms “polypeptide” and “peptide” will be usedinterchangeably to refer to a polymer of two or more amino acids joinedtogether by a peptide bond, wherein the peptide is of unspecifiedlength, thus, peptides, oligopeptides, polypeptides, and proteins areincluded within the present definition. In one aspect, this term alsoincludes post expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like. Includedwithin the definition are, for example, peptides containing one or moreanalogues of an amino acid or labeled amino acids and peptidomimetics.In a preferred embodiment, the present IBTs are comprised of L-aminoacids.

As used herein, the term “bioactive” or “peptide of interest activity”refers to the activity or characteristic associated with the peptideand/or protein of interest. The bioactive peptides may be used in avariety of applications including, but not limited to curative agentsfor diseases (e.g., insulin, interferon, interleukins, anti-angiogenicpeptides (U.S. Pat. No. 6,815,426), and polypeptides that bind todefined cellular targets (with the proviso that the peptide of interestis not an antibody or the F_(ab) fragment of an antibody) such asreceptors, channels, lipids, cytosolic proteins, and membrane proteins,to name a few), peptides having antimicrobial activity, peptides havingan affinity for a particular material (e.g., hair binding polypeptides,skin binding polypeptides, nail binding polypeptides, toothenamel-binding polypeptides, pellicle-binding peptides, cellulosebinding polypeptides, polymer binding polypeptides, clay bindingpolypeptides, silicon binding polypeptides, carbon nanotube bindingpolypeptides, and peptides that have an affinity for particular animalor plant tissues) for targeted delivery of benefit agents. The peptideof interest is typically no more than 300 amino acids in length,preferably less than 200 amino acids in length, and most preferably lessthan 100 amino acids in length. In a preferred embodiment, the peptideof interest is a peptide selected from a combinatorially generatedlibrary wherein the peptide is selected based on a specific affinity fora target substrate.

As used herein, the “benefit agent” refers to a molecule that imparts adesired functionality to a complex involving the peptide of interest fora defined application. The benefit agent may be a peptide of interestitself or may be one or more molecules bound to (covalently ornon-covalently), or associated with, the peptide of interest wherein thebinding affinity of the targeted polypeptide is used to selectivelytarget the benefit agent to the targeted material. In anotherembodiment, the targeted polypeptide comprises at least one regionhaving an affinity for at least one target material (e.g., biologicalmolecules, polymers, hair, skin, nail, clays, other peptides, etc.) andat least one region having an affinity for the benefit agent (e.g.,pharmaceutical agents, pigments, conditioners, dyes, fragrances, etc.).In another embodiment, the peptide of interest comprises a plurality ofregions having an affinity for the target material and a plurality ofregions having an affinity for the benefit agent. In yet anotherembodiment, the peptide of interest comprises at least one region havingan affinity for a targeted material and a plurality of regions having anaffinity for a variety of benefit agents wherein the benefit agents maybe the same of different. Examples of benefits agents may include, butare not limited to conditioners for personal care products, pigments,dyes, fragrances, pharmaceutical agents (e.g., targeted delivery ofcancer treatment agents), diagnostic/labeling agents, ultraviolet lightblocking agents (i.e., active agents in sunscreen protectants), andantimicrobial agents (e.g., antimicrobial peptides), to name a few.

“Codon degeneracy” refers to the nature in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment that encodes the present amino acidsequences. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a gene forexpression in a host cell, it is desirable to design the gene such thatits frequency of codon usage approaches the frequency of preferred codonusage of the host cell.

As used herein, the term “solubility” refers to the amount of asubstance that can be dissolved in a unit volume of a liquid underspecified conditions. In the present application, the term “solubility”is used to describe the ability of a peptide (inclusion body tag,peptide of interest, or fusion peptides) to be resuspended in a volumeof solvent, such as a biological buffer. In one embodiment, the peptidestargeted for production (“peptides of interest”) are normally soluble inthe cell and/or cell lysate under normal physiological conditions.Fusion of one or more inclusion body tags (IBTs) to the target peptideresults in the formation of a fusion peptide that is insoluble undernormal physiological conditions, resulting in the formation of inclusionbodies. In one embodiment, the peptide of interest is insoluble in anaqueous medium having a pH range of 5-12, preferably 6-10; and atemperature range of 5° C. to 50° C., preferably 10° C. to 40° C.

The term “amino acid” refers to the basic chemical structural unit of aprotein or polypeptide. The following abbreviations are used herein toidentify specific amino acids:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any naturally-occurringamino acid Xaa X (or as defined by the formulas described herein)

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences (includingcoding regions engineered to encode fusion peptides) that are not foundtogether in nature. Accordingly, a chimeric gene may comprise regulatorysequences and coding sequences that are derived from different sources,or regulatory sequences and coding sequences derived from the samesource, but arranged in a manner different than that found in nature. A“foreign” gene refers to a gene not normally found in the host organism,but that is introduced into the host organism by gene transfer. Foreigngenes can comprise native genes inserted into a non-native organism, orchimeric genes.

As used herein, the term “coding sequence” refers to a DNA sequence thatencodes for a specific amino acid sequence. “Suitable regulatorysequences” refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences may include promoters, enhancers, ribosomal binding sites,translation leader sequences, introns, polyadenylation recognitionsequences, RNA processing site, effector binding sites, and stem-loopstructures. One of skill in the art recognizes that selection ofsuitable regulatory sequences will depend upon host cell and/orexpression system used.

As used herein, the term “genetic construct” refers to a series ofcontiguous nucleic acids useful for modulating the genotype or phenotypeof an organism. Non-limiting examples of genetic constructs include butare not limited to a nucleic acid molecule, and open reading frame, agene, a plasmid and the like.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J. and Russell,D., Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and bySilhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with GeneFusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor, N.Y.(1984); and by Ausubel, F. M. et. al., Short Protocols in MolecularBiology, 5^(th) Ed. Current Protocols and John Wiley and Sons, Inc.,N.Y., 2002.

As used herein, the terms “fusion peptide”, “fusion protein”, “chimericprotein”, and “chimeric peptide” can be used interchangeably and referto a polymer of amino acids (peptide, oligopeptide, polypeptide, orprotein) comprising at least two portions, each portion comprising adistinct functionally independent peptide. In a fusion peptide wherein afirst peptide PEP1 and a second peptide, PEP2, are both directly andcovalently bound to an acid cleavable linker, L, through peptide bonds,a result of acid cleavage of the acid cleavable peptide linker, L, willbe to disrupt the covalent bond between PEP1 and PEP2 rendering themsoluble but no longer covalently joined. The result is that PEP1 andPEP2 would be separable by conventional biochemical methodology.

As used herein, the term “heterologous” refers to peptides andpolypeptides that are not naturally encoded by a cell's genome, but areprogrammed to be synthesized by a cell that has been recombinantlyengineered by standard gene transfer methods. As used herein, the termheterologous encompasses the fusion peptides of the invention, which areencoded by expression vectors that are introduced into the desired celltype. As used herein, a recombinant cell, a recombinant microbial cell,a recombinant yeast cell and a recombinant bacterial cell are cells thathave been genetically or recombinantly engineered to synthesize theheterologous fusion peptides of the invention. In some cases, thesynthesis of a heterologous peptide or polypeptide may be the result ofthe infection of a cell by either a eukaryotic virus or a prokaryoticbacteriophage.

In one embodiment, the invention encompasses a method of isolating apeptide of interest (“POI”) from a recombinant cell expressing aheterologous fusion peptide comprising the linker L. The recombinantcell may be any prokaryotic or eukaryotic cell type, including microbialcells. Preferred recombinant microbial cells include recombinant yeastand recombinant bacterial cells. An additional embodiment contemplatestaking advantage of the fact that heterologous peptide expression inrecombinant cells is often accompanied by the newly synthesizedheterologous fusion peptides accumulating in insoluble inclusion bodieswithin the nucleus or the cytoplasm of the cell. When such insolublefusion peptides having a POI also comprise the acid-cleavable linker L,it is contemplated herein that acid hydrolysis of L will liberate thePOI from the inclusion body, preferably to a more soluble form. Thereleased soluble form of the POI is then more easily separable fromremaining inclusion bodies and insoluble remnants thereof.

As used herein, an “inclusion body” is an insoluble intracellulardeposit of aggregated heterologous polypeptide(s) found in the cytoplasmor nucleus of a recombinant prokaryotic or eukaryotic cell. In apreferred embodiment the inclusion body is found in a recombinantmicrobial cell. In an even more preferred embodiment the recombinantmicrobial cell is a recombinant yeast cell or a recombinant bacterialcell. In a further preferred embodiment the recombinant bacterial cellis a recombinant Escherichia coli.

As used herein, the term “solubility tag” or “inclusion body tag,” i.e.,“IBT,” will refer to a peptide or polypeptide that facilitates formationof inclusion bodies when fused to a peptide of interest. The peptide ofinterest is preferably soluble within the host cell and/or host celllysate when not fused to an inclusion body tag. Fusion of the peptide ofinterest to an inclusion body tag produces a fusion protein thataccumulates into intracellular bodies (inclusion bodies) within the hostcell.

Peptides of interest that are typically soluble within the host celland/or cell lysates can be fused to one or more inclusion body tags tofacilitate formation of an insoluble fusion protein. In an alternativeembodiment, the peptide of interest may be partially insoluble in thehost cell, but produced at relatively lows levels where significantinclusion body formation does not occur. As such, the formation ofinclusion bodies will increase peptide production. In a furtherembodiment, fusion of the peptide of interest to one or more inclusionbody tags (IBTs) increases the amount of protein produced in the hostcell. Formation of the inclusion body facilitates simple and efficientpurification of the fusion peptide from the cell lysate using techniqueswell known in the art such as centrifugation and filtration. In anotherembodiment, the inclusion body tag comprises an effective number ofcross-linkable cysteine residues useful for separating the IBT from thepeptide of interest (post cleavage into a mixture of peptide fragments)with the proviso that the peptide of interest is devoid of cysteineresidues. The fusion protein typically includes one or more cleavablepeptide linkers used to separate the protein/polypeptide of interestfrom the inclusion body tag(s). The cleavable peptide linker is designedso that the inclusion body tag(s) and the protein/polypeptide(s) ofinterest can be easily separated by cleaving the linker element. Thepeptide linker can be cleaved chemically (e.g., acid hydrolysis) orenzymatically (i.e., use of a protease/peptidase that preferentiallyrecognizes an amino acid cleavage site and/or sequence within thecleavable peptide linker).

After the inclusion bodies are separated and/or partially-purified orpurified from the cell lysate, the cleavable linker elements can becleaved chemically and/or enzymatically to separate the inclusion bodytag from the peptide of interest. The fusion peptide may also include aplurality of regions encoding one or more peptides of interest separatedby one or more cleavable peptide linkers.

As used herein, “acid-cleavable linker,” “acid-labile linker,”“acid-cleavable peptide,” and “acid cleavage site” may be usedinterchangeably and refers to a peptide that displays at least oneacid-cleavable peptide bond under the conditions specified herein. Thelinker function of the acid-cleavable peptide is based on the fact thatthe linker is covalently bonded to at least two peptides, PEP1 and PEP2wherein the at least two peptides are either identical or distinct fromeach other. Cleaving the linker, L, results in the breaking of a peptidebond, e.g., the bond between an aspartic acid and a proline residue orbetween two proline residues. The result would be that PEP1 and PEP2would no longer be covalently joined.

In one embodiment, the acid-cleavable linker is bonded at either itscarboxy terminus or amino terminus to an inclusion body tag (“IBT”) anda peptide of interest (“POI”) at the opposite terminus. Schematically,this may be represented according to the structural formula PEP1-L-PEP2,where either PEP1 or PEP2 may be either an IBT or POI and L representsan acid-cleavable linker.

In a further embodiment of PEP1-L-PEP2, both PEP1 and PEP2 are POIs. Insuch an embodiment, the fusion peptide is likely to be soluble, as wouldbe the cleaved forms of PEP1 and PEP2. In an additional embodiment,wherein both PEP1 and PEP2 are POIs, it may unexpectedly arise that thefusion peptide is insoluble in the recombinant cell. This is likely toarise when either of the POIs unexpectedly functions as an IBT. In suchcases, the acid-cleavage and peptide isolation may proceed as in thecase where one of either PEP1 or PEP2 is a known IBT peptide.

In the context of the present invention, the term “isolate” or“isolated” refers to separating a given peptide or cellular component(e.g., inclusion body) from other cellular proteins, structures,components, debris, molecules, and the like, without any inference ofhaving achieved a specific degree of purity. For illustration purposes,isolating a fusion peptide, POI or an inclusion body may arise when amixture of components comprising a fusion peptide, POI or inclusion bodyis submitted to one or more process steps resulting in an enrichment ofthe fusion peptide, inclusion body or POI over the starting mixture ofcomponents. Isolating any given component separates it from some, butnot necessarily all components of a cell homogenate, lysate or extract.Put another way, the term “isolated” may refer to either a purifiedcomponent or a partially purified component. In the latter, no degree ofpurity should be inferred. Similarly, the term “separated” or“separating” and the like can be used interchangeably with “isolated”and “isolating,” as well as other similarly used terms.

In the context of the claimed invention the term “solution ofsufficiently acidic pH” refers to any aqueous or organic liquid having apH value that is sufficiently low to cleave the acid-cleavable linker L.These include organic solvents, water, or any saline, or bufferedsaline, or growth medium having a pH value that is sufficiently low tocleave the acid-cleavable linker L. A solution of sufficiently acidic pHencompasses solutions that are formulated to lower the intracellular pHof intact cells, the pH of the environment of disrupted or solubilizedcells, or any mixture of cellular components, in order to promote thecleavage of linker L within the fusion peptide PEP1-L-PEP2.

A “POI” (i.e., protein of interest) is any peptide having one or moreactivities or functions that render it of interest to persons ofordinary skill in the art. Accordingly, the POI can possess any kind offunctionality including, but not limited to, receptors, ligands,enzymes, diagnostic markers, cellular or viral structural components andthe like. The term “independently functional” indicates that a POI orIBT demonstrates its activities or functions without participation by,or interaction with, an additional component of the fusion peptidePEP1-L-PEP2. Thus, for example, after hydrolytic release from either asoluble or insoluble fusion peptide, a POI will demonstrate its relevantproperties, activities or functions under the proper conditions.

As used herein, a non-POI portion of the fusion peptide PEP1-L-PEP2 iswhat remains of the fusion peptide after the POI is removed by acidcleavage. Thus, in some instances, the non-POI portion of the fusionpeptide could be represented by the IBT alone, or the IBT fused to aportion of the cleaved linker L. When the fusion peptide is insoluble,the non-POI portion of the fusion peptide refers to the insolubleportion of the fusion peptide remaining after acid cleavage of linker L.

Acid Cleavable Linkers and Fusion Peptides

A shown in FIG. 1, multimers of the single acid-cleavable DP pairprovided enhanced rates of acid hydrolysis of an intact fusion peptideaccording to the order DP4>DP3>DP2>DP1 (see Table 2). Experiments inwhich individual amino acid substitutions were made at the positionimmediately following (i.e., on the carboxy side) the proline residue inthe DP pair indicated that adding the aspartic acid had little if anyeffect (FIG. 4). The only amino acid substitution at this position thatsignificantly enhanced the rate of acid hydrolysis was an additionalproline (FIG. 4). Thus, the resultant sequence DPP reduced the half-timeof acid hydrolysis by approximately two-fold over rate observed with theDP pair (FIG. 4).

Similar amino acid substitutions were made on the amino terminal side ofthe DP pair's aspartic acid residue (FIG. 3). While not as marked aneffect as in the previous experiment, proline on the amino terminus sideof the DP pair also had the shortest t_(1/2) of all amino acidsubstitutions. Of note is that the hydrophobic amino acids tryptophanand phenylalanine were actually significantly more inhibiting the acidhydrolysis than the other amino acids.

With this background, additional linkers were designed and prepared asindicated in Table 2. For illustration purposes only Table 2 provides anon-limiting number of embodiments of acid-cleavable peptide linkersthat can be achieved given the empirical observations disclosed herein.Given this background and the technical guidance disclosed hereinpersons of ordinary skill in the art would be able to add to the list ofacid-cleavable linkers that are encompassed by the inventive conceptdetailed in this specification and to the numerous uses for whichcleavable peptide linkers are generally known in the art. For examplethe invention is useful for the expression and recovery of recombinantlyproduced peptides and proteins. Such proteins typically have high valuein any number of applications including, but not limited to medical,biomedical, diagnostic, personal care, and affinity applications wherethe peptides of interest are used as linkers to various surfaces.

In one embodiment, the invention encompasses an acid-cleavable peptidelinker, L, selected from the group consisting of:

(SEQ ID NO: 1) A. DPDP, (SEQ ID NO: 2) B. DPDPDP, (SEQ ID NO: 3)C. DPDPDPDP, (SEQ ID NO: 4) D. DPDPDPP, (SEQ ID NO: 5) E. DPDPPDPP,(SEQ ID NO: 6) F. DPDPPDP, and (SEQ ID NO: 7) G. DPPDPPDP,

wherein D is aspartic acid and P is proline;

In a preferred embodiment, the acid-cleavable peptide linker, L, isselected from the group consisting of DPDPDPP (SEQ ID NO:4), DPDPPDPP(SEQ ID NO:5), DPDPPDP (SEQ ID NO:6), and DPPDPPDP (SEQ ID NO:7).

The enhancement in the rate of acid hydrolysis demonstrated by thelinkers of the invention is defined as the rate of acid hydrolysis of anintact fusion peptide comprising the acid-cleavable linker of theinvention, as compared to the rate of acid hydrolysis of a fusionpeptide when the linker comprises a single DP pair. In the context ofthis invention and throughout the specification, the acid-cleavablelinkers may be referred to in a short-hand notation. Table 2 provides anonlimiting illustrative list of specific embodiments of theacid-cleavable linkers showing their short-hand designations as well astheir sequences and SEQ ID NO:.

TABLE 2 Embodiments of Linker L Linker Amino Acid SEQ  Name SequenceID NO: DP2 DPDP 1 DP3 DPDPDP 2 DP4 DPDPDPDP 3 PP1 DPDPDPP 4 PP2 DPDPPDPP5 PP3 DPDPPDP 6 PP4 DPPDPPDP 7

An additional embodiment of the invention encompasses a fusion peptideor fusion polypeptide comprising the acid-cleavable peptide linkerwherein the fusion peptide has the structure,

PEP1-L-PEP2,

wherein PEP1 and PEP2 are functional peptides or polypeptides that arecovalently linked to one another through the acid-cleavable peptidelinker L.

In an additional embodiment of the invention, a fusion peptide or fusionpolypeptide consisting essentially of one of the present acid-cleavablepeptide linkers is provided wherein the fusion peptide has thestructure,

PEP1-L-PEP2,

wherein PEP1 and PEP2 are functional peptides or polypeptides that arecovalently linked to one another through the acid-cleavable peptidelinker L.

Either PEP1 or PEP2, or both, may be a peptide of interest (“POI”). Suchan embodiment of the fusion peptide may be soluble or insoluble in thecytoplasm of a recombinant cell. In the case wherein PEP1 and PEP2 arePOIs that confer cytoplasmic solubility to the fusion peptide, thefusion peptide may be isolated from cellular components or even purifiedprior to acidic cleavage of linker L. Thus, the acid-cleavable linkersand fusion peptide of the present invention provide suitable means forthe separation of a POI from a soluble fusion peptide.

As a nonlimiting illustrative example, the POI (e.g., PEP1) may be fusedthrough L to an antigenic peptide (e.g., PEP2) to which specificantibodies are known. Conventional methodology thereby allows the fusionpeptide to be isolated by immunological means, e.g., affinitychromatography on a column comprising an immobilized antibody directedto the antigenic peptide, as a first isolation step. Then aftersubsequent acid hydrolysis of the purified fusion peptide, the antigenicpeptide may be separated from the POI by again submitting theacid-treated mixture to another round of affinity chromatography orimmunoadsorption; e.g., on an affinity column or otheraffinity-substrate. Thus, the fusion peptide PEP1-L-PEP2 is suitablyversatile to aid in the purification of POIs from soluble fusionpeptides as well as from insoluble inclusion bodies.

As an additional nonlimiting illustrative example, the POI (e.g., PEP1)may be fused through L to a metal ion binding domain (e.g., PEP2) forwhich methods of adsorption to an ion-containing substrate areincorporated to purify the fusion peptide. Examples of suchmetal-binding domains include the 6×-His tag, or the metallothioneinpolypeptide or zinc-binding fragment thereof. Persons of ordinary skillin the art will recognize that these methods can be adapted to manysituations wherein the newly synthesized fusion peptide remains solublein the cell cytoplasm and PEP2, i.e. the non-POI portion of the fusionpeptide is a known ligand or receptor that can be isolated by adsorptionto an appropriate ligand-containing or receptor-containing substrate orsupport.

In an additional embodiment, one of PEP1 or PEP2 is a POI whereas theother of PEP1 or PEP2 is an inclusion body tag (“IBT”). In the contextof this description an IBT functions to direct newly synthesized fusionpeptide molecules into insoluble inclusion bodies within the recombinantcell, e.g., bacterial cell cytoplasm. In this embodiment, theacid-cleavable peptide linker provides a relatively simple means torelease a POI from the insoluble portion of the fusion peptidecomprising the IBT. For example, one could prepare an isolatedpreparation of inclusion bodies containing the desired fusion peptidewherein either of PEP1 or PEP2 is a POI with the remaining peptide beingan IBT. Resuspending, contacting, or incubating the inclusion bodies inan acidic medium of sufficiently low pH and at the appropriatetemperature for sufficient time would cleave the acid-cleavable peptidelinker joining PEP1 to PEP2, thereby selectively cleaving the linkerthereby yielding the POI in a soluble form while the IBT (or non-POIportion of the fusion peptide) remains insoluble. Therefore, the ease ofseparating the released soluble POI from the insoluble inclusion bodiesprovides a convenient and effective peptide purification step that canbe combined with conventional biochemical peptide isolation methodology.

An additional type of fusion peptide may arise fortuitously,specifically wherein either of PEP1 or PEP2 unexpectedly acts to directthe newly synthesized fusion peptide into inclusion bodies. Statedanother way, the situation may arise where a fusion peptide having aspecific combination of PEP1 and PEP2, neither of which was known tohave IBT-like properties, may unexpectedly accumulate in inclusionbodies. As long as at least one of either PEP1 or PEP2 becomes solubleafter acid cleavage, isolation of a POI can be achieved according to themethods disclosed herein.

The acid-cleavable linkers within the fusion peptide cleave at an acidicpH of between about pH 1 and about pH 6, preferably between a pH ofabout pH 1 and about pH 4, more preferably between about pH 2 and aboutpH 4, even more preferably between about pH 3 and about pH 4, and mostpreferably about pH 4. Thus, it follows that the fusion peptide of thepresent invention would also be expected to be cleaved within the samepH ranges.

The enhanced rates of acid hydrolysis of the linkers and fusion peptidesof the present invention are evidenced at a range of temperatures at theappropriate pH. Suitable temperature ranges are between about 40° C. toabout 90° C., preferably from about 50° C. to about 80° C., morepreferably between about 60° C. to about 70° C., and most preferablyabout 60° C.

In a further embodiment, the acid-cleavable linker is cleaved byincubating the fusion peptide at a pH of about pH 2 to about pH 4 and ata temperature of about 50° C. to about 80° C.

In view of this description, an even further embodiment of the inventionencompassed herein comprises a method of preparing at least one peptideof interest (“POI”) from a fusion peptide comprising at least one POI,comprising:

a) providing a recombinant cell synthesizing a fusion peptide having thestructure

PEP1-L-PEP2

-   -   wherein,    -   i) PEP1 and PEP2 are independently functional peptides wherein        at least one is a peptide of interest (“POI”); and    -   ii) L is an acid-cleavable linker comprising a peptide selected        from the group consisting of:

(SEQ ID NO: 1) A. DPDP, (SEQ ID NO: 2) B. DPDPDP, (SEQ ID NO: 3)C. DPDPDPDP, (SEQ ID NO: 4) D. DPDPDPP, (SEQ ID NO: 5) E. DPDPPDPP,(SEQ ID NO: 6) F. DPDPPDP, and (SEQ ID NO: 7) G. DPPDPPDP,

wherein D is aspartic acid and P is proline;

b) contacting the fusion peptide with a solution of sufficiently acidicpH so that linker L is cleaved, and

c) isolating the at least one POI.

It is contemplated that the recombinant cell be either prokaryotic oreukaryotic. Preferably, the recombinant cell is a microbial cell, andmore preferably a recombinant bacterial cell. A preferred recombinantbacterial cell is a recombinant Escherichia coli cell.

The method of preparing the POI comprises an acid cleaving step that isperformed at an acidic pH of between about 1 and about 6, preferablybetween a pH of about 1 and about 4, and more preferably between about 2and about 3.

With respect to temperature, the acid cleaving step is performed at asuitable temperature range of between about 40° C. to about 90° C.,preferably from about 50° C. to about 80° C., and more preferablybetween about 50° C. or 60° C. to about 70° C.

For the purpose of practicing the inventive method inclusion bodies maybe isolated from recombinant cells using any known methods. The releaseof the POI from the insoluble IBT-containing complex can be affected inpreparations of inclusion bodies of varied purity. Therefore theinventive method may be used in conjunction with various additionalmethods of isolating inclusion bodies based on the specific needs ofpersons of ordinary skill in the art. In another embodiment, theinclusion bodies in whole recombinant cell homogenates or wholerecombinant cell extracts may be acid treated without further enrichmentor purification and still provide acid hydrolytic release of the POI toa soluble form that is separable from the insoluble remnant of thefusion peptide and the remaining inclusion bodies. In an even furtherembodiment, the invention encompasses a recombinant cell, morespecifically a recombinant yeast cell or recombinant bacterial cell,which expresses such a fusion peptide. In this context an especiallydesirable recombinant bacterial cell is Escherichia coll.

A still further embodiment of the invention is the isolated or purifiedinclusion bodies comprising a fusion peptide of interest. The inclusionbodies comprising the fusion peptide PEP1-L-PEP2 function as aconvenient means to store, freeze, transport POIs in a form from whichthey are easily separated from the unwanted portion by acid hydrolysisand isolated by conventional biochemical techniques.

Inclusion Body Tags

The fusion peptide comprising an IBT may further comprise an effectivenumber of cross-linkable cysteine residues. As described in co-pendingU.S. Provisional Patent Application No. 60/951,754 entitled “RecombinantPeptide Production Using a Cross-Linkable Solubility Tag”, the inclusionof an effective number of cross-linkable cysteine residues is useful toselectively precipitate and separate the IBT from the POI duringprocessing. Upon acidic cleavage of the fusion peptide, the mixture offragments (IBTs and POIs) is subjected to oxidizing conditions for aperiod of time sufficient to cross-link the effective number of cysteineresidues incorporated into the IBT. The oxidative cross-linkingselectively precipitates the IBTs from the soluble peptide of interestwith the proviso that the peptide of interest is devoid ofcross-linkable cysteine residues.

IBTs comprising cysteine residues may be effectively used as solubilitytags in combination with a peptide of interest having cross-linkablecysteine residues. However, in such situations an oxidative-crosslinking step will typically be omitted during subsequent POI isolation.

Peptides of Interest

The peptide of interest (“POI”) targeted for production using thepresent method is one that is appreciably soluble in the host celland/or host cell liquid lysate under normal physiological conditions. Ina preferred aspect, the peptides of interest are generally short (<300amino acids in length) and difficult to produce in sufficient amountsdue to proteolytic degradation. Fusion of the peptide of interest to atleast one of the present inclusion body forming tags creates a fusionpeptide that is insoluble in the host cell and/or host cell lysate undernormal physiological conditions. Production of the peptide of interestis typically increased when expressed and accumulated in the form of aninsoluble inclusion body as the peptide is generally more protected fromproteolytic degradation. Furthermore, the insoluble fusion protein canbe easily separated from the host cell lysate using centrifugation orfiltration.

In general, the inventive acid-cleavable linkers can be used in aprocess to produce any peptide of interest that is (1) typically solublein the cell and/or cell lysate under typical physiological conditionsand/or (2) those that can be produced at significantly higher levelswhen expressed in the form of an inclusion body. In a preferredembodiment, the peptide of interest is appreciably soluble in the hostcell and/or corresponding cell lysate under normal physiological and/orprocess conditions.

The length of the peptide of interest may vary as long as (1) thepeptide is appreciably soluble in the host cell and/or cell lysate,and/or (2) the amount of the targeted peptide produced is significantlyincreased when expressed in the form of an insoluble fusionpeptide/inclusion body (i.e. expression in the form of a fusion proteinprotect the peptide of interest from proteolytic degradation). Typicallythe peptide of interest is less than 300 amino acids in length,preferably less than 100 amino acids in length, more preferably lessthan 75 amino acids in length, even more preferably less than 50 aminoacids in length, and most preferably less than 25 amino acids in length.

The function of the peptide of interest is not limited by the presentmethod and may include, but is not limited to bioactive molecules suchas curative agents for diseases (e.g., insulin, interferon,interleukins, peptide hormones, anti-angiogenic peptides, and peptides(with the proviso that the peptide is not an antibody or an F_(ab)portion of an antibody) that bind to and affect defined cellular targetssuch as receptors, channels, lipids, cytosolic proteins, and membraneproteins; see U.S. Pat. No. 6,696,089,), peptides having an affinity fora particular material (e.g., biological tissues, biological molecules,hair-binding peptides (U.S. Patent Application Publication Nos.2005-0226839, 2003-0152976, and 2002-0098524; International PatentApplication Publication Nos. WO01/79479 and WO04/048399; and U.S. Pat.Nos. 7,736,633; 7,427,656; and 7,749,957), skin-binding peptides (U.S.Pat. Nos. 7,309,482; 7,427,656; 7,749,957; and 7,341,604), nail-bindingpeptides (U.S. Patent Application Publication No. 2005-0226839; U.S.Pat. No. 7,749,957), cellulose-binding peptides, polymer-bindingpeptides (U.S. Pat. Nos. 7,632,919; 7,928,076; 7,700,716; and7,906,617), and clay-binding peptides (U.S. Pat. No. 7,749,957), fortargeted delivery of at least one benefit agent (U.S. Pat. Nos.7,220,405 and 7,749,957; and U.S. Patent Application Publication No.2005-0226839).

In a preferred aspect, the peptide of interest is an affinity peptideidentified from a combinatorially generated peptide library. In afurther aspect, the peptide is selected from a combinatorially generatedlibrary wherein said library was prepared using a technique selectedfrom the group consisting of phage display, yeast display, bacterialdisplay, ribosomal display and mRNA display.

In a preferred aspect, the peptide of interest is selected from thegroup of hair binding peptides, skin binding peptides, nail bindingpeptides, tooth binding peptides, antimicrobial peptides, pigmentbinding peptides, clay-binding peptides, mineral binding peptides (e.g.,calcium carbonate), and various polymer binding peptides.

Affinity peptides are particularly useful to target benefit agentsimparting a desired functionality to a target material (e.g., hair,skin, etc.) for a defined application (U.S. Pat. Nos. 7,220,405;7,736,633; and 7,749,957; and U.S. Patent Application Publication No.2005-0226839 for a list of typical benefit agents such as conditioners,pigments/colorants, fragrances, etc.). The benefit agent may be peptideof interest itself or may be one or more molecules bound to (covalentlyor non-covalently), or associated with, the peptide of interest whereinthe binding affinity of the peptide of interest is used to selectivelytarget the benefit agent to the targeted material. In anotherembodiment, the peptide of interest comprises at least one region havingan affinity for at least one target material (e.g., biologicalmolecules, polymers, hair, skin, nail, other peptides, etc.) and atleast one region having an affinity for the benefit agent (e.g.,pharmaceutical agents, antimicrobial agents, pigments, conditioners,dyes, fragrances, etc.). In another embodiment, the peptide of interestcomprises a plurality of regions having an affinity for the targetmaterial and a plurality of regions having an affinity for one or morebenefit agents. In yet another embodiment, the peptide of interestcomprises at least one region having an affinity for a targeted materialand a plurality of regions having an affinity for a variety of benefitagents wherein the benefit agents may be the same of different. Examplesof benefits agents may include, but are not limited to conditioners forpersonal care products, pigments, dye, fragrances, pharmaceutical agents(e.g., targeted delivery of cancer treatment agents),diagnostic/labeling agents, ultraviolet light blocking agents (i.e.,active agents in sunscreen protectants), and antimicrobial agents (e.g.,antimicrobial peptides), to name a few.

Cleavable Peptide Linkers

Fusion peptides comprising inclusion body tags will typically include atleast one cleavable sequence separating the inclusion body tag from thepolypeptide of interest. The cleavable sequence facilitates separationof the inclusion body tag(s) from the peptide(s) of interest. In oneembodiment, the cleavable sequence may be provided by a portion of theinclusion body tag and/or the peptide of interest (e.g., inclusion of anacid cleavable aspartic acid-proline moiety). In a preferred embodiment,the cleavable sequence is provided by including (in the fusion peptide)at least one cleavable peptide linker between the inclusion body tag andthe peptide of interest.

Generally, means to cleave peptide linkers include chemical hydrolysis,enzymatic agents, and combinations thereof. In one embodiment, one ormore chemically cleavable peptide linkers are included in the fusionconstruct to facilitate recovery of the peptide of interest from theinclusion body fusion protein. Examples of chemical cleavage reagentsinclude cyanogen bromide (cleaves methionine residues), N-chlorosuccinimide, iodobenzoic acid or BNPS-skatole[2-(2-nitrophenylsulfenyl)-3-methylindole] (cleaves tryptophanresidues), dilute acids (cleaves at aspartic acid-proline bonds), andhydroxylamine (cleaves at asparagine-glycine bonds at pH 9.0); seeGavit, P. and Better, M., J. Biotechnol., 79:127-136 (2000); Szoka etal., DNA, 5(1):11-20 (1986); and Walker, J. M., The Proteomics ProtocolsHandbook, 2005, Humana Press, Totowa, N.J.)).

In a preferred embodiment, one or more aspartic acid-prolineacid-cleavable recognition sites (i.e., a cleavable peptide linkercomprising one or more D-P dipeptide moieties) are included in thefusion protein construct to facilitate separation of the inclusion bodytag(s) from the peptide of interest.

In another embodiment, the fusion peptide may include multiple regionsencoding peptides of interest separated by one or more cleavable peptidelinkers.

In another embodiment, one or more enzymatic cleavage sequences areincluded in the fusion protein construct to facilitate recovery of thepeptide of interest. Examples of enzymes useful for cleaving the peptidelinker may include, but are not limited to Arg-C proteinase, Asp-Nendopeptidase, chymotrypsin, clostripain, enterokinase, Factor Xa,glutamyl endopeptidase, Granzyme B, Achromobacter proteinase I, pepsin,proline endopeptidase, proteinase K, Staphylococcal peptidase I,thermolysin, thrombin, trypsin, and members of the Caspase family ofproteolytic enzymes (e.g. Caspases 1-10) (Walker, J. M., supra). Anexample of a cleavage site sequence is the Caspase-3 cleavage site(Thornberry et al., J. Biol. Chem., 272:17907-17911 (1997) and Tyas etal., EMBO Reports, 1(3):266-270 (2000)).

Typically, the cleavage step occurs after the insoluble inclusion bodiesand/or insoluble fusion peptides are isolated from the cell lysate. Thecells can be lysed using any number of means well known in the art (e.g.mechanical and/or chemical lysis). Methods to isolate the insolubleinclusion bodies/fusion peptides from the cell lysate are well known inthe art (e.g., centrifugation, filtration, and combinations thereof).Once recovered from the cell lysate, the insoluble inclusion bodiesand/or fusion peptides can be treated with a cleavage agent (chemical orenzymatic) to cleavage the inclusion body tag from the peptide ofinterest. In one embodiment, the fusion protein and/or inclusion body isdiluted and/or dissolved in a suitable solvent prior to treatment withthe cleavage agent. In a further embodiment, the cleavage step may beomitted if the inclusion body tag does not interfere with the activityof the peptide of interest.

After the cleavage step, and in a preferred embodiment, the peptide ofinterest can be separated and/or isolated from the fusion protein andthe inclusion body tags based on a differential solubility of thecomponents. Parameters such as pH, salt concentration, and temperaturemay be adjusted to facilitate separation of the inclusion body tag fromthe peptide of interest. In one embodiment, the peptide of interest issoluble while the inclusion body tag and/or fusion protein is insolublein the defined process medium (typically an aqueous medium). In anotherembodiment, the peptide of interest is insoluble while the inclusionbody tag is soluble in the defined process medium.

In a preferred embodiment, the inclusion body tag comprises an effectivenumber of cross-linkable cysteine residues with the proviso that thepeptide of interest is devoid of cysteine residues (U.S. Pat. No.7,951,559). Upon cleavage, oxidative cross-linking is used toselectively cross-link the IBTs (typically insoluble). The conditionsare controlled so that the cross-linked IBTs are insoluble while thepeptide of interest remains soluble. The soluble peptide of interest issubsequently separated from the cross-linked IBTs using a conventionalseparation techniques such as centrifugation.

In an optional embodiment, the peptide of interest may be furtherpurified using any number of purification techniques in the art such asion exchange, gel purification techniques, and column chromatography(see U.S. Pat. No. 5,648,244), to name a few.

Fusion Peptides

Inclusion body tags are used to create chimeric polypeptides (“fusionpeptides” or “fusion proteins”) that are insoluble within the host cell,forming inclusion bodies. Methods of synthesis and expression of geneticconstructs encoding the present fusion peptides is well known to one ofskill in the art. The present fusion peptides will include at least oneinclusion body tag

(IBT) functionally linked to at least one peptide of interest (POI) viaan acid-cleavable linker of the present invention. Typically, the fusionpeptides will also include at least one cleavable peptide linker havinga cleavage site between the inclusion body tag and the peptide ofinterest. In one embodiment, the inclusion body tag may include acleavage site whereby inclusion of a separate cleavable peptide linkermay not be necessary. In a preferred embodiment, the cleavage method ischosen to ensure that the peptide of interest is not adversely affectedby the cleavage agent(s) employed. In a further embodiment, the peptideof interest may be modified to eliminate possible cleavage sites withthe peptide so long as the desired activity of the peptide is notadversely affected.

One of skill in the art will recognize that the elements of the fusionprotein can be structured in a variety of ways. Typically, the fusionprotein will include at least one IBT (i.e., PEP1), at least one peptideof interest (POI) (i.e., PEP2), and at least one cleavable peptidelinker (L) located between the IBT and the POI. Thus, such a fusionpeptide conforms to the general structure PEP1-L-PEP2. The inclusionbody tag may be organized as a leader sequence or a terminator sequencerelative to the position of the peptide of interest within the fusionpeptide. In another embodiment, a plurality of IBTs, POIs, and Ls areused when engineering the fusion peptide. In a further embodiment, thefusion peptide may include a plurality of IBTs (as defined herein),POIs, and Ls that are the same or different.

In another embodiment of the fusion peptide, neither of PEP1 or PEP2comprises an IBT, but rather the fusion peptide remains soluble. As anonlimiting illustrative, example, the POI (e.g., PEP1) may be fusedthrough L to an antigenic peptide (e.g., PEP2) to which specificantibodies are known. Conventional methodology thereby allows the fusionpeptide to be isolated by immunological means, e.g., affinitychromatography on a column comprising an immobilized antibody directedto the antigenic peptide, as a first isolation step. Then aftersubsequent acid hydrolysis of the purified fusion peptide, the antigenicpeptide may be separated from the POI by again submitting theacid-treated mixture to another round of affinity chromatography orimmunoadsorption on; e.g., an affinity column or otheraffinity-substrate. Thus, the fusion peptide PEP1-L-PEP2 is suitablyversatile to aid in the purification of POIs from soluble fusionpeptides as well as from insoluble inclusion bodies.

The fusion peptide should be insoluble in an aqueous medium at atemperature of about 10° C. to about 50° C., preferably about 10° C. toabout 40° C. The aqueous medium typically comprises a pH range of aboutpH 5 to about pH 12, preferably about pH 6 to about pH 10, and mostpreferably about pH 6 to about pH 8. The temperature, pH, and/or ionicstrength of the aqueous medium can be adjusted to obtain the desiredsolubility characteristics of the fusion peptide/inclusion body.

Method of Making a Peptides of Interest Using Insoluble Fusion Peptides

The inclusion body tags are used to make fusion peptides that forminclusion bodies within the production host. This method is particularlyattractive for producing significant amounts of soluble peptide ofinterest that (1) are difficult to isolation from other solublecomponents of the cell lysate and/or (2) are difficult to product insignificant amounts within the target production host.

In the present methods, a POI is fused to one end of an inventiveacid-cleavable linker while an IBT is fused at the other end therebyforming an insoluble fusion protein. Expression of the genetic constructencoding the fusion protein produces an insoluble form of the peptide ofinterest that accumulates in the form of inclusion bodies within thehost cell. The host cell is grown for a period of time sufficient forthe insoluble fusion peptide to accumulate within the cell.

The host cell is subsequently lysed using any number of techniques wellknown in the art. The insoluble fusion peptide/inclusion bodies are thenseparated from the soluble components of the cell lysate using a simpleand economical technique such as centrifugation and/or membranefiltration. The insoluble fusion peptide/inclusion body can then befurther processed in order to isolate the peptide of interest.Typically, this will include resuspension of the fusionpeptide/inclusion body in a liquid medium suitable for cleaving thefusion peptide, separating the inclusion body tag from the peptide ofinterest. The fusion protein is typically designed to include acleavable peptide linker separating the inclusion body tag from thepeptide of interest. The cleavage step can be conducted using any numberof techniques well known in the art (chemical cleavage, enzymaticcleavage, and combinations thereof). The peptide of interest can then beseparated from the inclusion body tag(s) and/or fusion peptides usingany number of techniques well known in the art (centrifugation,filtration, precipitation, column chromatography, etc.). Preferably, thepeptide of interest (once cleaved from fusion peptide) has a solubilitythat is significantly different than that of the inclusion body tagand/or remaining fusion peptide. In a further preferred embodiment,oxidative cross-linking is used to selectively precipitate the IBT(comprising an effective number of cross-linkable cysteine residues)from the peptide of interest (when devoid of cross-linkable cysteineresidues). For example, IBT139.CCPGCC, IBT-139(5C), and IBT186, weredesigned to include an effective number of cross-linkable cysteineresidues.

Transformation and Expression

Once the inclusion body tag has been identified and paired with theappropriate peptide of interest, construction of cassettes and vectorsthat may be transformed in to an appropriate expression host is commonand well known in the art. Typically, the vector or cassette containssequences directing transcription and translation of the relevantchimeric gene, a selectable marker, and sequences allowing autonomousreplication or chromosomal integration. Suitable vectors comprise aregion 5′ of the gene which harbors transcriptional initiation controlsand a region 3′ of the DNA fragment which controls transcriptionaltermination. It is most preferred when both control regions are derivedfrom genes homologous to the transformed host cell, although it is to beunderstood that such control regions need not be derived from the genesnative to the specific species chosen as a production host.

Transcription initiation control regions or promoters, which are usefulto drive expression of the genetic constructs encoding the fusionpeptides in the desired host cell, are numerous and familiar to thoseskilled in the art. Virtually any promoter capable of driving theseconstructs is suitable for the present invention including but notlimited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1,URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1(useful for expression in Pichia); and lac, ara (pBAD), tet, trp,IP_(L), IP_(R), T7, tac, and trc (useful for expression in Escherichiacoli) as well as the amy, apr, npr promoters and various phage promotersuseful for expression in Bacillus.

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary; however, it is most preferred if included.

Preferred host cells for expression of the present fusion peptides aremicrobial hosts that can be found broadly within the fungal or bacterialfamilies and which grow over a wide range of temperature, pH values, andsolvent tolerances. For example, it is contemplated that any ofbacteria, yeast, and filamentous fungi will be suitable hosts forexpression of the present nucleic acid molecules encoding the fusionpeptides. Because of transcription, translation, and the proteinbiosynthetic apparatus is the same irrespective of the cellularfeedstock, genes are expressed irrespective of the carbon feedstock usedto generate the cellular biomass. Large-scale microbial growth andfunctional gene expression may utilize a wide range of simple or complexcarbohydrates, organic acids and alcohols (i.e. methanol), saturatedhydrocarbons such as methane or carbon dioxide in the case ofphotosynthetic or chemoautotrophic hosts. However, the functional genesmay be regulated, repressed or depressed by specific growth conditions,which may include the form and amount of nitrogen, phosphorous, sulfur,oxygen, carbon or any trace micronutrient including small inorganicions. In addition, the regulation of functional genes may be achieved bythe presence or absence of specific regulatory molecules that are addedto the culture and are not typically considered nutrient or energysources. Growth rate may also be an important regulatory factor in geneexpression. Examples of host strains include, but are not limited tofungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces,Pichia, Yarrowia, Candida, Hansenula, or bacterial species such asSalmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium,Erythrobacter, Chlorobium, Chromatium, Flavobacterium, Cytophaga,Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria,Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas,Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus,Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis,Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella, andMyxococcus. Preferred bacterial host strains include Escherichia,Pseudomonas, and Bacillus. In a highly preferred aspect, the bacterialhost strain is Escherichia coli.

Fermentation Media

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include but are not limited tomonosaccharides such as glucose and fructose, oligosaccharides such aslactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Additionally the carbon substrate may also be one-carbonsubstrates such as carbon dioxide, or methanol for which metabolicconversion into key biochemical intermediates has been demonstrated. Inaddition to one and two carbon substrates methylotrophic organisms arealso known to utilize a number of other carbon containing compounds suchas methylamine, glucosamine and a variety of amino acids for metabolicactivity. For example, methylotrophic yeast are known to utilize thecarbon from methylamine to form trehalose or glycerol (Bellion et al.,Microb. Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32. Editor(s):Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK).Similarly, various species of Candida will metabolize alanine or oleicacid (Sulter et al., Arch. Microbiol. 153:485-489 (1990)). Hence it iscontemplated that the source of carbon utilized in the present inventionmay encompass a wide variety of carbon containing substrates and willonly be limited by the choice of organism.

Although it is contemplated that all of the above mentioned carbonsubstrates and mixtures thereof are suitable in the present invention,preferred carbon substrates are glucose, fructose, and sucrose.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for the growthof the cultures and promotion of the expression of the present fusionpeptides.

Culture Conditions

Suitable culture conditions can be selected dependent upon the chosenproduction host. Typically, cells are grown at a temperature in therange of about 25° C. to about 40° C. in an appropriate medium. Suitablegrowth media may include common, commercially-prepared media such asLuria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast medium(YM) broth. Other defined or synthetic growth media may also be used andthe appropriate medium for growth of the particular microorganism willbe known by one skilled in the art of microbiology or fermentationscience. The use of agents known to modulate catabolite repressiondirectly or indirectly, e.g., cyclic adenosine 2′:3′-monophosphate, mayalso be incorporated into the fermentation medium.

Suitable pH ranges for the fermentation are typically between pH 5.0 topH 9.0, where pH 6.0 to pH 8.0 is preferred.

Fermentations may be performed under aerobic or anaerobic conditionswhere aerobic conditions are generally preferred.

Industrial Batch and Continuous Fermentations

A classical batch fermentation is a closed system where the compositionof the medium is set at the beginning of the fermentation and notsubject to artificial alterations during the fermentation. Thus, at thebeginning of the fermentation the medium is inoculated with the desiredorganism or organisms, and fermentation is permitted to occur withoutadding anything to the system. Typically, a “batch” fermentation isbatch with respect to the addition of carbon source and attempts areoften made at controlling factors such as pH and oxygen concentration.In batch systems the metabolite and biomass compositions of the systemchange constantly up to the time the fermentation is stopped. Withinbatch cultures cells moderate through a static lag phase to a highgrowth log phase and finally to a stationary phase where growth rate isdiminished or halted. If untreated, cells in the stationary phase willeventually die. Cells in log phase generally are responsible for thebulk of production of end product or intermediate.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch fermentation processes are also suitable in the presentinvention and comprise a typical batch system with the exception thatthe substrate is added in increments as the fermentation progresses.Fed-Batch systems are useful when catabolite repression is apt toinhibit the metabolism of the cells and where it is desirable to havelimited amounts of substrate in the media. Measurement of the actualsubstrate concentration in Fed-Batch systems is difficult and istherefore estimated on the basis of the changes of measurable factorssuch as pH, dissolved oxygen and the partial pressure of waste gasessuch as CO₂. Batch and Fed-Batch fermentations are common and well knownin the art and examples may be found in Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, Second Edition(1989) Sinauer Associates, Inc., Sunderland, Mass. (hereinafter“Brock”), or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227(1992).

Although the present invention is typically performed in batch mode itis contemplated that the method would be adaptable to continuousfermentation methods. Continuous fermentation is an open system where adefined fermentation medium is added continuously to a bioreactor and anequal amount of conditioned media is removed simultaneously forprocessing. Continuous fermentation generally maintains the cultures ata constant high density where cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or end product concentration.For example, one method will maintain a limiting nutrient such as thecarbon source or nitrogen level at a fixed rate and allow all otherparameters to moderate. In other systems a number of factors affectinggrowth can be altered continuously while the cell concentration,measured by media turbidity, is kept constant. Continuous systems striveto maintain steady state growth conditions and thus the cell loss due tothe medium being drawn off must be balanced against the cell growth ratein the fermentation. Methods of modulating nutrients and growth factorsfor continuous fermentation processes as well as techniques formaximizing the rate of product formation are well known in the art ofindustrial microbiology and a variety of methods are detailed by Brock,supra.

It is contemplated that the present invention may be practiced usingeither batch, fed-batch or continuous processes and that any known modeof fermentation would be suitable.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and the guidance provided by the Examples, oneskilled in the art can ascertain the essential characteristics of thisinvention, and without departing from the scope thereof, can makevarious changes and modifications of the invention to adapt it tovarious uses and conditions.

The meaning of abbreviations used is as follows: “min” means minute(s),“h” means hour(s), “μL” means microliter(s), “mL” means milliliter(s),“L” means liter(s), “nm” means nanometer(s), “mm” means millimeter(s),“cm” means centimeter(s), “μm” means micrometer(s), “mM” meansmillimolar, “M” means molar, “mmol” means millimole(s), “μmol” meansmicromole(s), “pmol” means picomole(s), “g” means gram(s), “μg” meansmicrogram(s), “mg” means milligram(s), “g” means the gravitationconstant, “rpm” means revolutions per minute, “DTT” meansdithiothreitol, and “cat#” means catalog number.

General Methods

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J. and Russell,D., Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and bySilhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with GeneFusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor, N.Y.(1984); and by Ausubel, F. M. et. al., Short Protocols in MolecularBiology, 5^(th) Ed. Current Protocols and John Wiley and Sons, Inc.,N.Y., 2002.

Materials and methods suitable for the maintenance and growth ofbacterial cultures are also well known in the art. Techniques suitablefor use in the following Examples may be found in Manual of Methods forGeneral Bacteriology, Phillipp Gerhardt, R. G. E. Murray, Ralph N.Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. BriggsPhillips, eds., American Society for Microbiology, Washington, D.C.,1994, or in Brock (supra). All reagents, restriction enzymes andmaterials used for the growth and maintenance of bacterial cells wereobtained from BD Diagnostic Systems (Sparks, Md.), Invitrogen (Carlsbad,Calif.), Life Technologies (Rockville, Md.), QIAGEN (Valencia, Calif.)or Sigma-Aldrich Chemical Company (St. Louis, Mo.), unless otherwisespecified.

Expression Vector pLD001

Plasmid pLD001 (SEQ ID NO: 254) has been previous reported as a suitableexpression vector for E. coli (see U.S. Patent Application PublicationNo. 2010-0158823 A1 to Wang et al.; incorporated herein by reference).

The vector pLD001 was derived from the commercially available vectorpDEST17 (Invitrogen, Carlsbad, Calif.). It includes sequences derivedfrom the commercially available vector pET31b (Novagen, Madison, Wis.)that encode a fragment of the enzyme ketosteroid isomerase (KSI). TheKSI fragment was included as a fusion partner to promote partition ofthe peptides into insoluble inclusion bodies in E. coli. TheKSI-encoding sequence from pET31 b was modified using standardmutagenesis procedures (QuickChange II, Stratagene, La Jolla, Calif.) toinclude three additional Cys codons, in addition to the one Cys codonfound in the wild type KSI sequence. In addition, all Asp codons in thecoding sequence were replaced by Glu codons. Plasmid pLD001, given bySEQ ID NO: 254, was constructed using standard recombinant DNA methods,which are well known to those skilled in the art.

Coding sequences bounded by BamHI and AscI sites may be ligated betweenBamHI and AscI sites in pLD001 using standard recombinant DNA methods.The resulting gene fusions resulted in a peptide of interest was fuseddownstream from a modified fragment of ketosteroid isomerase (KSI(C4)E)that served to drive the peptide into insoluble inclusion bodies in E.coli (See U.S. Patent Application Publication No. 2009-0029420A1; hereinincorporated by reference)

Example 1 Preparation of Plasmid pLX121

A genetic construct was prepared for evaluating the performance of theinventive acid-cleavable linker by fusing the linker to an inclusionbody tag on one end, and a soluble peptide of interest at the linker'sopposite end. The peptide of interest used in the present examples wasprepared from a previously reported peptide-based triblock dispersant(U.S. Patent Application Publication No. 2005-0054752).

Cloning of the TBP1 Gene

The TBP1 gene, encoding the TBP1 peptide, was selected for evaluation ofthe inventive acid-cleavable linkers. The synthetic TBP1 peptide ispeptide-based triblock dispersant comprising a carbon-black bindingdomain, a hydrophilic peptide linker, and a cellulose binding domain(see Example 15 of U.S. patent application Ser. No. 10/935,254, hereinincorporated by reference).

The TBP1 gene (SEQ ID NO: 17) encoding the 68 amino acid peptide TBP101(SEQ ID NO: 19) was assembled from synthetic oligonucleotides(Sigma-Genosys, Woodlands, Tex.; Table 3).

TABLE 3 Oligonucleotides Used to Prepare the TBP1 Oligonucleotide SEQ Name Nucleotide Sequence (5′-3′) ID NO: TBP1(+)1GGATCCATCGAAGGTCGTTTCCACGAA  8 AACTGGCCGTCTGGTGGCGGTACCTCTACTTCCAAAGCTTCCACCACTACGAC TTCTAGCAAAACCACCACTACAT TBP1(+)2CCTCTAAGACTACCACGACTACCTCCAA  9 AACCTCTACTACCTCTAGCTCCTCTACGGGCGGTGGCACTCACAAGACCTCTACTC AGCGTCTGCTGGCTGCATAA TBP1(−)1TTATGCAGCCAGCAGACGCTGAGTAGAG 10 GTCTTGTGAGTGCCACCGCCCGTAGAGGAGCTAGAGGTAGT TBP1(−)2 AGAGGTTTTGGAGGTAGTCGTGGTAGTC 11TTAGAGGATGTAGTGGTGGTTTTGCTAG AAGTCGTAGTGGT TBP1(−)3GGAAGCTTTGGAAGTAGAGGTACCGC 12 CACCAGACGGCCAGTTTTCGTGGAAACGACCTTCGATGGATCC

Each oligonucleotide was phosphorylated with ATP using T4 polynucleotidekinase. The resulting oligonucleotides were mixed, boiled for 5 min, andthen cooled to room temperature slowly. Finally, the annealedoligonucleotides were ligated with T4 DNA ligase to give synthetic DNAfragment TBP1, given as SEQ ID NO: 17, which encodes the TBP101 peptide(SEQ ID NO: 19).

Construction of pINK101 Expression Plasmid:

Lambda phage site-specific recombination was used for preparation andexpression of the present fusion proteins (Gateway™ System; Invitrogen,Carlsbad, Calif.). TBP1 was integrated into the Gateway™ system forprotein over-expression. In the first step, 2 μL of the TBP1 ligationmixture was used in a 50-μL PCR reaction. Reactions were catalyzed byPfu DNA polymerase (Stratagene, La Jolla, Calif.), following thestandard PCR protocol. Primer 5′TBP1 (5′-CACCGGATCCATCGAAGGTCGT-3′; SEQID NO: 21) and 3′TBP1 (5′-TCATTATGCAGCCAGCAGCGC-3′; SEQ ID NO: 20) wereused for amplification of the TBP1 fragment. The design of these primersadds an additional sequence of CACC and another stop codon TGA wereadded to the 5′ and 3′ ends of the amplified fragments.

The amplified TBP1 was directly cloned into pENTR™/D-TOPO® vector (SEQID NO: 22) using Invitrogen's pENTR™ directional TOPO® cloning kit(Invitrogen; Catalog K2400-20), resulting in the Gateway™ entry plasmidpENTR-TBP1. This entry plasmid was propagated in One Shot® TOP10 E. colicells (Invitrogen). The accuracy of the PCR amplification and cloningprocedures were confirmed by DNA sequencing analysis. The entry plasmidwas mixed with pDEST17 (Invitrogen, SEQ ID NO: 23). LR recombinationreactions were catalyzed by LR CLONASE™ (Invitrogen). The destinationplasmid, pINK101 was constructed and propagated in the DH5a E. colistrain. The accuracy of the recombination reaction was determined by DNAsequencing. All reagents for LR recombination reactions (i.e., lambdaphage site-specific recombination) were provided in Invitrogen's E. coliexpression system with the GATEWAY™ Technology kit. The site-specificrecombination process followed the manufacturer's instructions(Invitrogen).

The resulting plasmid, named pINK101, contains the coding region forrecombinant protein 6H-TBP1, named INK101 (SEQ ID NO 18), which is an11.6 kDa protein. The protein sequence includes a 6×His tag and a 24amino acid linker that includes Factor Xa protease recognition sitebefore the sequence of the TBP101 peptide.

The amino acid coding region for the 6×His tag and the following linkercomprising the Factor Xa protease recognition site were excised frompINK101 by digestion with the NdeI and BamHI restriction enzymes.

The TBP1 gene (SEQ ID NO:17) encodes a polypeptide (SEQ ID NO:19) havinga ST linker flanked by Gly-Gly-Gly amino acids. The system was made moremodular by further mutagenesis to change the upstream amino acidsequence from Gly-Gly-Gly to Ala-Gly-Gly (codon GGT changed to GCC) andthe downstream Gly-Gly-Gly to Gly-Gly-Ala (codon GGT GGC changed to GGCGCC). These changes provided a NgoMI restriction site and a Kaslrestriction site flanking the ST linker, thus facilitating replacementof any element in TBP1.

Further modifications were made to TBP101 including the addition of anacid cleavable site to facilitate the removal of any tag sequenceencoded by the region between the NdeI and BamHI sites of the expressionplasmid. The resulting plasmid was called pLX121 (also referred to as“pINK101DP”; SEQ ID NO: 250). These modifications changed the aminoacids E-G to D-P (acid cleavable aspartic acid-proline linkage) usingthe Stratagene QUIKCHANGE® II Site-Directed Mutagenesis Kit Cat#200523(La Jolla, Calif.) as per the manufacturer's protocol using the primersINK101+(5′-CCCCTTCACCGGATCCATCGATCCACGTTTCCACGAAAACTGGCC-3′; SEQ ID NO:24) and INK101-(5′-GGCCAGTTTTCGTGGAAACGTGGATCGATGGATCCGGTGAAGGGG-3′; SEQID NO: 25). The sequences were confirmed by DNA sequence analysis. Thecoding region (SEQ ID NO: 251) and the corresponding amino acid sequenceof the modified protein (SEQ ID NO: 26), INK101DP, are provided.

INK101DP Peptide (SEQ ID NO: 26):MSYYHHHHHHLESTSLYKKAGSAAAPFTGSIDPRFHENWPSAGGTSTSKASTTTTSSKTTTTSSKTTTTTSKTSTTSSSSTGGATHKTSTQRLLAAThe aspartic acid-proline acid cleavable linker is in bold type. The DPpair replaces the EG pair found in the unmodified TBP101 peptide. Themodified TBP101 peptide (i.e., a model peptide of interest) isunderlined.

The INK101DP peptide conforms to the general structure PEP1-L-PEP2,wherein PEP1 containing the 6×His tag and Factor Xa cleavage site wasfound to function as an IBT, whereas PEP2 functions as a POI (i.e., acarbon black binding peptide). In this instance, the 6×His tag was foundto be necessary for directing the accumulation of the newly synthesizedfusion peptide in insoluble inclusion bodies within the recombinantbacterial cell cytoplasm.

Example 2 Acid Hydrolysis of Peptide Linkers Containing Multiple DPResidues

This example demonstrates the enhanced rate of acid hydrolysis of afusion peptide having a linker comprising more than one consecutivelyarranged aspartic acid-proline (i.e., DP) pair. Specifically, the rateof acid hydrolysis of fusion peptides having peptide linkers comprisingone, two, three or four consecutive DP pairs were measured and compared.

Strain and Media

Escherichia coli BL21-A1 was obtained from Invitrogen Corp. (Cat.#607003, Carlsbad, Calif.). Expression plasmid pINK101DP (Example 1) waspreviously described in U.S. Patent Application Publication No.2005-0054752) Cells were grown at 37° C. in Miller's LB broth (Cat.#46-050-CM, Mediatech, Inc., Herndon, Va.) with 0.2% L-(+)-arabinose(Cat. # A3256, Sigma-Aldrich, Inc., St. Louis, Mo.) and 100 μg/mLampicillin (Cat. # A1066, Sigma-Aldrich, Inc., St. Louis, Mo.). Cellswere plated on LB agar plates with 100 μg/mL ampicillin (Cat. # L1004,Teknova, Inc., Hollister, Calif.).

Construction of pINK101DP Variants Containing Additional DPs

One to three additional DP residues were added to pINK101DP bysite-directed mutagenesis using a QUIKCHANGE™ II Kit (Cat. #200524,Stratagene, La Jolla, Calif.). Primers pairs used to add additional DPresidues are provided in Table 4. Reactions were thermocycled in a GeneAmp 9700 using the thermocycling parameters provided in Table 5 (PerkinElmer Applied Biosystems, Norwalk, Conn.). Escherichia coli BL21-A1 wastransformed with 1 μL of QUIKCHANGE™ reaction product according tomanufacturer's directions and transformants were selected on LB agarplates with 100 μg/mL ampicillin. DNA sequences were obtained for sixisolates from each transformation in order to identify those with thedesired mutations.

TABLE 4 Mutagenesis Primers Primer SEQ  ID Nucleotide Sequence ID NO:for DP2 CCCCTTCACCGGATCCATCGATCCAG 13 ATCCACGTTTCCACGAAAACTGGCC for DP3CCCCTTCACCGGATCCATCGATCCAG 14 ATCCAGATCCACGTTTCCACGAAAAC TGGCC for DP4CCCCTTCACCGGATCCATCGATCCAG 15 ATCCAGATCCAGATCCACGTTTCCAC GAAAACTGGCCRemote GTAATACGGTTATCCACAGAATCAG 16 Rev

Reaction Components—

10X QUIKCHANGE ™ reaction buffer 5 μL QUIKCHANGE ™ dNTP mix 1 μL Pfupolymerase 1 μL 10 μM forward primer 1 μL 10 μM reverse primer 1 μLWater 40 mL

TABLE 5 Thermocycling program Segment Cycles Temperature Time 1 1 95° C.2 minutes 2 18 95° C. 50 seconds 55° C. 1 minute 68° C. 10 minutes 3 168° C. 10 minutes 4 1  4° C. hold

Preparation of Peptide-Containing Inclusion Bodies

Strains containing each mutant plasmid were grown for 18 hrs at 37° C.in LB with 0.2% arabinose and 100 μg/mL ampicillin. Cells were lysed byadding 75 mg/mL CELYTIC™ Express reagent (Cat. # C1990, Sigma Aldrich,St. Louis, Mo.) and incubating at 37° C. for 20 minutes. Inclusion bodypellets were separated from lysed cell cultures by centrifugation at9,000×g for 1 minute. Pellets were washed three times with ⅓^(rd) theoriginal culture volume of 20 mM Tris-CI, pH 8.0, and then resuspendedin 1/10^(th) the original culture volume of 20 mM Tris-CI, pH8.0.

Acid Hydrolysis of Peptide in Inclusion Body Pellets

Pellets were washed once and then resuspended in 1/10^(th) the originalculture volume of sterile, filtered water. One mL of inclusion bodysuspension was pelleted by centrifugation at 9,000×g for 1 minute andthen resuspended in 500 μL of 20 mM H₂PO₄, pH 2.2 (Cat. #0260-1, J.T.Baker, Phillipsburg, N.J.). A 100 μL time-zero sample was removed andneutralized by adding 50 μL of 100 mM MES, pH 8.9 (Cat. #475893,Calbiochem, La Jolla, Calif.). The remaining inclusion body sample wasincubated at 65° C. Additional samples were taken at 2, 6 and 24 hoursand neutralized in the same manner as the time-zero sample.

Separation of Hydrolyzed Peptide/Tag Fragments

25 μL of each neutralized sample was mixed with 75 μL 8 M guanidine-HCl(Cat. #5502UA, Life Technologies, Inc., Gaithersburg, Md.) and 25 μL ofthat mixture was injected onto a GraceVydacC18 HPLC column (Cat.#218TP54, Resolution Systems, Holland Mich.) run on an Agilent 1100 HPLCsystem (Agilent, Foster City, Calif.). Run conditions were 0.1% TFA inwater and acetonitrile, gradient from 10-90% acetonitrile in 28 minutes,0.35 mL/min, 40° C. Peak identities were confirmed by running fractionson denaturing acrylamide gels.

Analysis of HPLC Data

HPLC data was analyzed using ChemStation software (Hewlett Packard GmbH,Waldbrunn, Germany). Hydrolysis rates were determined by measuring thereduction in peak area for the unhydrolyzed peptide at each time point(FIG. 1).

Results and Conclusion

The rate of acid hydrolysis of the acid-labile linkers was based on therate of disappearance of the corresponding parent fusion peptide (FIG.1). Generally, each fusion peptide comprising more than one DP pair perlinker was acid cleaved at a higher rate than the fusion peptide withone DP pair thereby indicating the relative rates of acid-cleavagedemonstrated by the linkers of the present invention. In general, therates of acid hydrolysis of the fusion peptides having the correspondinglinkers follows the order DP4>DP3>DP2>DP.

The rate of acid-cleavage of the fusion peptide can be modeled underthis set of conditions as a simple linear combination of first orderrate constants (FIG. 2). Decreasing the t_(1/2) for hydrolysis can beaccomplished by increasing the DP number.

In the experiments described in Example 4, linkers comprising threeconsecutive DP pairs, i.e. DP3, formed the core linker into whichadditional proline residues were introduced at various locations withinthe DP3 sequence. The introduction of one or more additional prolineresidues immediately after the proline of DP pair was predicated on theresults of saturation mutagenesis experiments described in Example 3,and summarized in FIGS. 3 and 4. These experiments show that anadditional proline added immediately after the proline of a single DPpair, provided enhanced fusion peptide hydrolysis rates.

Example 3 Effect of Saturation Mutagenesis at Amino Acid PositionsImmediately Preceding or Following a Single DP Linker

This Example demonstrates the effects of amino acid changes in thepositions immediately upstream of the D residue in a single DP pair(i.e., the IBT side of the test fusion peptide) and downstream of the Presidue in the single DP pair (i.e., the POI side of the test fusionpeptide).

Construction of pINK101DP Variants Containing Amino Acid Changes onEither Side of DP Linker

The strains and media follow those described in Example 2. Multiplechanges were made to residues on either side of the DP linker inpINK101DP by site-directed mutagenesis using a QUIKCHANGE™ II Kit (Cat.#200524, Stratagene, La Jolla, Calif.). Reactions were thermocycled in aGene Amp 9700 (Perkin Elmer Applied Biosystems, Norwalk, Conn.). Theprimers used to introduce changes are provided in Table 6 and thethermocycling program parameters are provided in Table 7. Escherichiacoli BL21-A1 was transformed with 1 μL of QUIKCHANGE™ reaction productaccording to manufacturer's directions and transformants were selectedon LB agar plates with 100 μg/mL ampicillin. In cases where notransformants were obtained (DP-Asp, DP-Glu, DP-Gly, DP-His, DP-Ile,DP-Leu, DP-Lys, DP-Met, DP-Pro, DP-Thr, DP-Trp), 2 μL of QUIKCHANGE™reaction product was used to transform E. coli 10G EliteElectrocompetent Cells (Lucigen Corp., Middleton, Wis.) according tomanufacturer's directions, for the purpose of generating super-coiledplasmid DNA. Transformants were selected on LB agar plates with 100μg/mL ampicillin. DNA sequences were obtained for six isolates from eachtransformation in order to identify those with the desired mutations.Plasmid was prepared from the identified isolates using QIAprep SpinMiniprep Kit (QIAGEN Inc., Valencia, Calif.). Escherichia coli BL21-A1was then transformed with 10 ng of plasmid DNA according tomanufacturer's directions and transformants were selected on LB agarplates with 100 μg/mL ampicillin.

TABLE 6 Primers used to introduce modifications to residues flanking DP residues Primer SEQ  ID Nucleotide SequenceID NO: DP1Alafor CACCGGATCCATCGATCCAGCATT 27 CCACGAAAACTGGCCGTCDP1Alarev GACGGCCAGTTTTCGTGGAATGCT 28 GGATCGATGGATCCGGTG DP1AsnforCACCGGATCCATCGATCCAAACTT 29 CCACGAAAACTGGCCGTC DP1AsnrevGACGGCCAGTTTTCGTGGAAGTTT 30 GGATCGATGGATCCGGTG DP1AspforCACCGGATCCATCGATCCAGATTT 31 CCACGAAAACTGGCCGTC DP1AsprevGACGGCCAGTTTTCGTGGAAATCT 32 GGATCGATGGATCCGGTG DP1CysforCACCGGATCCATCGATCCATTGTT 33 CCACGAAAACTGGCCGTC DP1CysrevGACGGCCAGTTTTCGTGGAACAAT 34 GGATCGATGGATCCGGTG DP1GlnforCACCGGATCCATCGATCCACAGTT 35 CCACGAAAACTGGCCGTC DP1GlnrevGACGGCCAGTTTTCGTGGAACTGT 36 GGATCGATGGATCCGGTG DP1GluforCACCGGATCCATCGATCCAGAATT 37 CCACGAAAACTGGCCGTC DP1GlurevGACGGCCAGTTTTCGTGGAATTCT 38 GGATCGATGGATCCGGTG DP1GlyforCACCGGATCCATCGATCCAGGATT 39 CCACGAAAACTGGCCGTC DP1GlyrevGACGGCCAGTTTTCGTGGAATCCT 40 GGATCGATGGATCCGGTG DP1HisforCACCGGATCCATCGATCCACACTT 41 CCACGAAAACTGGCCGTC DP1HisrevGACGGCCAGTTTTCGTGGAAGTGT 42 GGATCGATGGATCCGGTG DP1IleforCACCGGATCCATCGATCCAATCTT 43 CCACGAAAACTGGCCGTC DP1IlerevGACGGCCAGTTTTCGTGGAAGATT 44 GGATCGATGGATCCGGTG DP1LeuforCACCGGATCCATCGATCCACTCTT 45 CCACGAAAACTGGCCGTC DP1LeurevGACGGCCAGTTTTCGTGGAAGAGT 46 GGATCGATGGATCCGGTG DP1LysforCACCGGATCCATCGATCCAAAATT 47 CCACGAAAACTGGCCGTC DP1LysrevGACGGCCAGTTTTCGTGGAATTTT 48 GGATCGATGGATCCGGTG DP1MetforCACCGGATCCATCGATCCAATGTT 49 CCACGAAAACTGGCCGTC DP1MetrevGACGGCCAGTTTTCGTGGAACATT 50 GGATCGATGGATCCGGTG DP1PheforCACCGGATCCATCGATCCATTCTT 51 CCACGAAAACTGGCCGTC DP1PherevGACGGCCAGTTTTCGTGGAAGAAT 52 GGATCGATGGATCCGGTG DP1ProforCACCGGATCCATCGATCCACCATT 53 CCACGAAAACTGGCCGTC DP1ProrevGACGGCCAGTTTTCGTGGAATGGT 54 GGATCGATGGATCCGGTG DP1SerforCACCGGATCCATCGATCCATCCTT 55 CCACGAAAACTGGCCGTC DP1SerrevGACGGCCAGTTTTCGTGGAAGGAT 56 GGATCGATGGATCCGGTG DP1ThrforCACCGGATCCATCGATCCAACCTT 57 CCACGAAAACTGGCCGTC DP1ThrrevGACGGCCAGTTTTCGTGGAAGGTT 58 GGATCGATGGATCCGGTG DP1TrpforCACCGGATCCATCGATCCATGGTT 59 CCACGAAAACTGGCCGTC DP1TrprevGACGGCCAGTTTTCGTGGAACCAT 60 GGATCGATGGATCCGGTG DP1TyrforCACCGGATCCATCGATCCATACTT 61 CCACGAAAACTGGCCGTC DP1TyrrevGACGGCCAGTTTTCGTGGAAGTAT 62 GGATCGATGGATCCGGTG DP1ValforCACCGGATCCATCGATCCAGTTTT 63 CCACGAAAACTGGCCGTC DP1ValrevGACGGCCAGTTTTCGTGGAAAACT 64 GGATCGATGGATCCGGTG AlaDP1forCCCCTTCACCGGATCCGCCGATCC 65 ACGTTTCCACGAAAAC ArgDP1forCCCCTTCACCGGATCCCGTGATCC 66 ACGTTTCCACGAAAAC AsnDP1forCCCCTTCACCGGATCCAACGATCC 67 ACGTTTCCACGAAAAC AspDP1forCCCCTTCACCGGATCCGATGATCC 68 ACGTTTCCACGAAAAC CysDP1forCCCCTTCACCGGATCCTTGGATCC 69 ACGTTTCCACGAAAAC GlnDP1forCCCCTTCACCGGATCCCAGGATCC 70 ACGTTTCCACGAAAAC GluDP1forCCCCTTCACCGGATCCGAAGATCC 71 ACGTTTCCACGAAAAC GlyDPlforCCCCTTCACCGGATCCGGAGATCC 72 ACGTTTCCACGAAAAC HisDP1forCCCCTTCACCGGATCCCACGATCC 73 ACGTTTCCACGAAAAC LeuDP1forCCCCTTCACCGGATCCCTCGATCC 74 ACGTTTCCACGAAAAC LysDP1forCCCCTTCACCGGATCCAAAGATCC 75 ACGTTTCCACGAAAAC MetDP1forCCCCTTCACCGGATCCATGGATCC 76 ACGTTTCCACGAAAAC PheDPlforCCCCTTCACCGGATCCTTCGATCC 77 ACGTTTCCACGAAAAC ProDP1forCCCCTTCACCGGATCCCCAGATCC 78 ACGTTTCCACGAAAAC SerDP1forCCCCTTCACCGGATCCTCCGATCC 79 ACGTTTCCACGAAAAC ThrDP1forCCCCTTCACCGGATCCACCGATCC 80 ACGTTTCCACGAAAAC TrpDP1forCCCCTTCACCGGATCCTGGGATCC 81 ACGTTTCCACGAAAAC TyrDP1forCCCCTTCACCGGATCCTACGATCC 82 ACGTTTCCACGAAAAC ValDP1forCCCCTTCACCGGATCCGTTGATCC 83 ACGTTTCCACGAAAAC AlaDP1revGTTTTCGTGGAAACGTGGATCGGC 84 GGATCCGGTGAAGGGG ArgDP1revGTTTTCGTGGAAACGTGGATCACG 85 GGATCCGGTGAAGGGG AsnDP1revGTTTTCGTGGAAACGTGGATCGTT 86 GGATCCGGTGAAGGGG AspDP1revGTTTTCGTGGAAACGTGGATCATC 87 GGATCCGGTGAAGGGG CysDP1revGTTTTCGTGGAAACGTGGATCCAA 88 GGATCCGGTGAAGGGG GlnDP1revGTTTTCGTGGAAACGTGGATCCTG 89 GGATCCGGTGAAGGGG GluDP1revGTTTTCGTGGAAACGTGGATCTTC 90 GGATCCGGTGAAGGGG GlyDP1revGTTTTCGTGGAAACGTGGATCTCC 91 GGATCCGGTGAAGGGG HisDP1revGTTTTCGTGGAAACGTGGATCGTG 92 GGATCCGGTGAAGGGG LeuDP1revGTTTTCGTGGAAACGTGGATCGAG 93 GGATCCGGTGAAGGGG LysDP1revGTTTTCGTGGAAACGTGGATCTTT 94 GGATCCGGTGAAGGGG MetDP1revGTTTTCGTGGAAACGTGGATCCAT 95 GGATCCGGTGAAGGGG PheDP1revGTTTTCGTGGAAACGTGGATCGAA 96 GGATCCGGTGAAGGGG ProDP1revGTTTTCGTGGAAACGTGGATCTGG 97 GGATCCGGTGAAGGGG SerDP1revGTTTTCGTGGAAACGTGGATCGGA 98 GGATCCGGTGAAGGGG ThrDP1revGTTTTCGTGGAAACGTGGATCGGT 99 GGATCCGGTGAAGGGG TrpDP1revGTTTTCGTGGAAACGTGGATCCCA 100 GGATCCGGTGAAGGGG TyrDP1revGTTTTCGTGGAAACGTGGATCGTA 101 GGATCCGGTGAAGGGG ValDP1revGTTTTCGTGGAAACGTGGATCAAC 102 GGATCCGGTGAAGGGG

PCR Reaction Components

10X QUIKCHANGE ™ reaction buffer 5 μL QUIKCHANGE ™ dNTP mix 1 μL Pfupolymerase 1 μL 10 μM forward primer 1 μL 10 μM reverse primer 1 μLWater 40 mL

TABLE 7 Thermocycling program parameters Segment Cycles Temperature Time1 1 95° C. 30 seconds 2 25 95° C. 30 seconds 55° C. 1 minute 68° C. 6minutes 3 1 68° C. 8 minutes 4 1  4° C. hold

Preparation of Peptide-Containing Inclusion Bodies

The preparation of peptide-containing inclusion bodies followed theprocedures described in Example 2.

Acid Hydrolysis of Peptide in Inclusion Body Pellets

Pellets were washed once and then resuspended in 1/10^(th) the originalculture volume of sterile, filtered water. 300 μL of inclusion bodysuspension was pelleted by centrifugation at 9,000×g for 1 minute andthen resuspended in 130 μL of 20 mM H₂PO₄, pH 2.2 (Cat. #0260-1, J.T.Baker, Phillipsburg, N.J.). A 40-μL time-zero sample was removed andneutralized by adding 20 μL of 100 mM MES, pH 8.9 (Cat. #475893,Calbiochem, La Jolla, Calif.). The remaining inclusion body sample wasincubated at 70° C. Additional samples were taken at 2 and 6 hours andneutralized in the same manner as the time-zero sample.

Separation of Hydrolyzed Peptide/Tag Fragments and HPLC Analysis

The hydrolyzed peptide/solubility tag fragments were separated using theprocess described in Example 2. HPLC analysis was conducted as describedin Example 2.

Results and Conclusion

The rate of hydrolysis was minimally affected by the majority of aminoacid changes immediately preceding the D residue of the DP pair in thelinker although proline in this position marginally enhanced thehydrolysis rate (FIG. 3). Substitution of tryptophan or phenylalaninefor isoleucine on the amino-terminal side of the DP linker significantlyslows the rate of acid hydrolysis (FIG. 3). As such, substitutingtryptophan or phenylalanine on the amino-terminal side of the DP linkermay be useful to increase the stability of the DP linker in applicationswhere acid cleavage of a DP pair is not desired (e.g., a DP linker ispresent in a protein or peptide of interest where acid cleavage is notdesired).

An unexpected result was that only the substitution of proline forarginine at the position immediately following the proline residue ofthe DP linker substantially increases the rate of acid hydrolysis (FIG.4). Therefore, the effect on acid hydrolysis rates of additional prolineresidues inserted upon a DP3 background was assessed.

Example 4 Effect of Introducing Additional Proline Residues to theDPDPDP Linker

Based on the conclusion from Example 3, that an additional proline onthe C-terminal side of the single DP linker further accelerates the rateof hydrolysis, one or two prolines were added to an analogous positionwithin the DP3 linker. Example 4 demonstrates the effect on acidhydrolysis rate of the test fusion peptide of adding proline residues tothe DPDPDP linker (SEQ ID NO:2). These derivatives of DP3 are referredto as PP1, PP2, PP3 and PP4 (see Table 2 for sequences).

The strain, growth media, and construction of expression plasmid pDP3 isdescribed in Example 2.

Construction of pDP3 Variants Containing Additional Proline Residues inthe DP3 Linker

Additional proline residues were introduces at various positions in theDP3 linker by site-directed mutagenesis using a QUIKCHANGE™ II Kit (Cat.#200524, Stratagene, La Jolla, Calif.). The primers used to prepare thecorresponding variants are shown in Table 8. Reactions were thermocycledin a Gene Amp 9700 (Perkin Elmer Cetus, Norwalk, Conn.). The PCRreaction components and the thermocycling program parameters followthose described in Example 3. Escherichia coli BL21-A1 was transformedwith 1 μL of QUIKCHANGE™ reaction product according to manufacturer'sdirections and transformants were selected on LB agar plates with 100μg/mL ampicillin. Transformants with the desired mutations wereidentified by DNA sequencing. In the case of PP4, no transformants wereobtained and the transformation was repeated using E. coli 10G EliteElectrocompetent Cells as described in Example 3.

TABLE 8 Primers used in the construction of DP3 variants SEQ IDPrimer ID Nucleotide Sequence NO: DPDPDPPforCATCGATCCAGATCCAGATCCACCACGTTT 104 “PP1” CCACGAAAACTGGCC DPDPDPPrevGGCCAGTTTTCGTGGAAACGTGGTGGATC 105 “PP1” TGGATCTGGATCGATG DPDPPDPPforCATCGATCCAGATCCACCAGATCCACCAC 106 “PP2” GTTTCCACGAAAACTGGC DPDPPDPPrevGCCAGTTTTCGTGGAAACGTGGTGGATCT 107 “PP2” GGTGGATCTGGATCGATG DPDPPDPforGATCCATCGATCCAGATCCACCAGATCCAC 108 “PP3” GTTTCCACGAAAAC DPDPPDPrevGTTTTCGTGGAAACGTGGATCTGGTGGAT 109 “PP3” CTGGATCGATGGATC DPPDPPDPforCACCGGATCCATCGATCCACCAGATCCAC 110 “PP4” CAGATCCACGTTTCCACGAAAACDPPDPPDPrev GTTTTCGTGGAAACGTGGATCTGGTGGAT 111 “PP4”CTGGTGGATCGATGGATCCGGTG

Preparation of Peptide-Containing Inclusion Bodies

The preparation of the peptide-containing inclusion bodies follows theprocedures described in Example 2.

Acid Hydrolysis of Peptide in Inclusion Body Pellets

Pellets were washed once and then resuspended in 1/10^(th) the originalculture volume of sterile, filtered water. For PP1, 250 μL of inclusionbody suspension was pelleted by centrifugation at 9,000×g for 1 minuteand then resuspended in 120 μL of 20 mM H₂PO₄. For PP2, PP3, and PP4,500 μL of inclusion body suspension was pelleted by centrifugation at9,000×g for 1 minute and then resuspended in 120 μL of 20 mM H₂PO₄, pH2.2 (Cat. #0260-1, J.T. Baker, Phillipsburg, N.J.). A 20 μL time-zerosample was removed and neutralized by adding 10 μL of 100 mM MES, pH 8.9(Cat. #475893, Calbiochem, La Jolla, Calif.). The remaining inclusionbody sample was incubated at 70° C. Additional samples were taken at0.5, 1, 2 4 hours and neutralized in the same manner as the time-zerosample.

Separation of Hydrolyzed Peptide/Tag Fragments and HPLC Analysis

The hydrolyzed peptide/solubility tag fragments were separated using theprocess described in Example 2. HPLC analysis was conducted as describedin Example 2.

Results and Conclusion

The rate of hydrolysis is increased by the addition of proline residuesto the DP3 linker, with PP2 and PP4 showing the highest rate ofacid-cleavage (FIGS. 5 and 6).

Example 5 Effect of pH and Temperature on Acid Hydrolysis

The following example was conducted to demonstrate the effect of changesin pH and temperature on the rate of acid hydrolysis on various DPlinkers.

Strains and Media

Strains are described in Examples 2 and 4, media and growth conditionsin Example 2.

Preparation of Peptide-Containing Inclusion Bodies

The preparation of the peptide-containing inclusion bodies follows theprocedures described in Example 2.

Acid Hydrolysis of Peptide in Inclusion Body Pellets (Varying pH)

Pellets were washed once and then resuspended in 1/10^(th) the originalculture volume of sterile, filtered water. For DP1, DP3, and PP2; 580μL, 290 μL, and 720 μL of inclusion body suspension, respectively, waspelleted by centrifugation at 9,000×g for 1 minute and then resuspendedin 360 μL of sterile, filtered water. Each sample was divided into three120 μL aliquots and then re-pelleted by centrifugation at 9,000×g for 1minute. For each sample, one of the three aliquots was resuspended in0.25% phosphoric acid, pH 2.10 (Cat. #0260-1, J.T. Baker, Phillipsburg,N.J.), 0.25% formic acid, pH 2.44 (Cat. # FX0440-7, E. M. Science,Gibbstown, N.J.) or 0.25% acetic acid, pH 2.88 (Cat. # AX0073-6, EMDChemicals, Gibbstown, N.J.). A 20 μL time-zero sample was removed fromeach and neutralized by adding 10 μL of 100 mM MES, pH 8.9 (Cat.#475893, Calbiochem, La Jolla, Calif.). The remaining inclusion bodysample was incubated at 70° C. Additional samples were taken at 1, 2, 4and 6 hours and neutralized in the same manner as the time-zero sample.The results are provided in FIG. 7.

Acid Hydrolysis of Peptide in Inclusion Body Pellets (ReducedTemperature)

Pellets were washed once and then resuspended in 1/10^(th) the originalculture volume of sterile, filtered water. For DP3 and PP2, 240 μL and450 μL of inclusion body suspension, respectively, was pelleted bycentrifugation at 9,000×g for 1 minute and then resuspended in 240 μL ofsterile, filtered water. Each sample was divided into two 120 μLaliquots and then re-pelleted by centrifugation at 9,000×g for 1 minute.For each sample, one of the two aliquots was resuspended in 0.25%phosphoric acid, pH 2.10 (Cat. #0260-1, J.T. Baker, Phillipsburg, N.J.)or 0.25% formic acid, pH 2.44 (Cat. # FX0440-7, E. M. Science,Gibbstown, N.J.). A 20 μL time-zero sample was removed from each andneutralized by adding 10 μL of 100 mM MES, pH 8.9 (Cat. #475893,Calbiochem, La Jolla, Calif.). The remaining inclusion body sample wasincubated at 50° C. Additional samples were taken at 1, 2, 6 and 24hours and neutralized in the same manner as the time-zero sample. Theresults are provided in FIG. 8.

Separation of Hydrolyzed Peptide/Tag Fragments and HPLC Analysis

The hydrolyzed peptide/solubility tag fragments were separated using theprocess described in Example 2. HPLC analysis was conducted as describedin Example 2.

Results and Conclusion

The results of hydrolysis in 0.25% w/v acid solutions at 70° C. (FIG. 7)and 50° C. are illustrated (FIG. 8).

The relative hydrolysis rate in formic acid, pH 2.44, was notsubstantially different from phosphoric acid, pH 2.10 for either DP3 orPP2 at either 70° C. (FIG. 7) or 50° C. However, the hydrolysis rate at50° C. was reduced approximately 2-fold for PP2 and 3-fold for DP3 ascompared to the rate at 70° C.

Example 6 Preparation of Constructs Incorporating Different Acid LabileSequences

This example describes the assembly of four constructs that containdifferent acid-labile sequences that separate the two domains of theprotein. The fusion peptides were designed to have an inclusion body tag(KSI(C4E); SEQ ID NO: 252) linked to a peptide of interest (HC353; SEQID NO: 253) where the two components are separated by an acid-cleavablepeptide linker. The following four acid-cleavable linkers wereengineered between KSI(C4E) and HC353:

DP (SEQ ID NO: 2) DPDPDP (SEQ ID NO: 5) DPDPPDPP (SEQ ID NO: 7) DPPDPPDP

Construction of KSI(C4E).DP.HC353:

The gene for HC353 was synthesized by DNA2.0 (Menlo Park, Calif.) withBamHI and AscI flanking the 5′ and 3′ ends of the gene, respectively.The gene incorporated nucleotides that code for the amino acid sequenceDP just downstream of the BamHI site. The HC353 gene was cloned into theBamHI—AscI sites of the plasmid pLD001 (SEQ ID NO: 254), yieldingplasmid pJZ353.

The resulting DNA construct (SEQ ID NO: 255) encoded the fusion peptideKSI(C4E).DP.HC353 (SEQ ID NO: 256).

Construction of KSI(C4E).DPDPDP.HC353:

In order to modify the acid-cleavable linker between KSI(C4E) and HC353,two additional DP sequences were incorporated into the sequence encodingKSI(C4E).DP.HC353. This was accomplished by the QuikChange Site-DirectedMutagenesis kit by Stratagene (La Jolla, Calif.). The following twoprimers were used to introduce the additional sequences:

353.DP3 UP: (SEQ ID NO: 257) gcttgtcagggatccgatcctgaccctgatccatctgctcaatctcaactgcc 353.DP3 DOWN: (SEQ ID NO: 258)ggcagttgagattgagcagatggatcagggtcaggatcgg atccctgacaagcThe resulting plasmid pLR688 expressed a DNA construct (SEQ ID NO: 259)encoding fusion peptide KSI(C4E).DPDPDP.HC353 (SEQ ID NO: 260).

Construction of KSI(C4E).DPDPPDPP.HC353:

In order to modify the acid-cleavable linker between KSI(C4E) and HC353,two additional P residues were incorporated into the sequence encodingKSI(C4E).DPDPDP.HC353 (pLR688). This was accomplished by the QuikChangeSite-Directed Mutagenesis kit by Stratagene (La Jolla, Calif.). Thefollowing two primers were used to introduce the additional sequences:

PP2 HC353 UP: (SEQ ID NO: 261)gatccgatcctgaccctccagatccaccgtctgctcaatctcaactgc PP2 HC353 DOWN:(SEQ ID NO: 262) gcagttgagattgagcagacggtggatctggagggtcaggatcggatcThe resulting plasmid pLR726 expressed a DNA construct (SEQ ID NO: 263)encoding fusion peptide KSI(C4E).DPDPPDPP.HC353 (SEQ ID NO: 264).

Construction of KSI(C4E).DPPDPPDP.HC353:

In order to modify the acid-cleavable linker between KSI(C4E) and HC353,two additional P residues were incorporated into the sequence encodingKSI(C4E).DPDPDP.HC353 (pLR688). This was accomplished by the QuikChangeSite-Directed Mutagenesis kit by Stratagene (La Jolla, Calif.). Thefollowing two primers were used to introduce the additional sequences:

353 PP4 UP: (SEQ ID NO: 265)gtcagggatccgatcctccagaccctccagatccatctgctcaatc 353 PP4 DOWN:(SEQ ID NO: 266) gattgagcagatggatctggagggtctggaggatcggatccctgacThe resulting plasmid pLR816 expressed a DNA construct (SEQ ID NO: 267)encoding fusion peptide KSI(C4E).DPPDPPDP.HC353 (SEQ ID NO: 268).

Example 7 Production of IBT Fusions to Peptides HC353

Strains expressing the fusions of KSI(C4E) to peptide HC353 withvariants of the DP acid cleavage described in Example 6 were grown inone liter of autoinduction medium (10 g/L Tryptone, 5 g/L Yeast Extract,5 g/L NaCl, 50 mM Na₂HPO₄, 50 mM KH₂PO₄, 25 mM (NH₄)₂SO₄, 3 mM MgSO₄,0.75% glycerol, 0.075% glucose and 0.05% arabinose, 50 mg/mLSpectinomycin) at 37° C., and 200 rpm incubator shaker for 20 hours. Thecells were harvested by centrifugation, resuspended in 200 mL of lysisbuffer (50 mM Tris pH 7.5, 5 mM EDTA,100 mM NaCl) using a tissuehomogenizer (Brinkman Homogenizer model PCU11 at setting 3-4) andcollected again by centrifugation at 8000 rpm for 5 min. The washedcells were resuspended with a homogenizer in 200 mL of lysis buffer tothe pellet containing 50 mg of lysozyme and allowed to sit on ice forthree hours before being frozen at −20° C. The cell suspension wasthawed at 37° C., homogenized and subject to sonication using a BransonSonifier model 450 sonicator equipped with a 5 mm probe (BransonUltrasonics Corporation, Danbury, Conn.) at 20% maximum output, 2 pulsesper second for 1 min, repeat once.

The disrupted cells were transferred to 50-mL conical tubes and theinsoluble fraction containing the inclusion bodies was harvested bycentrifugation for 10 min at 10,000 rpm. The inclusion bodies wereresuspended in 200 mL of BENZONASE® buffer (20 mM Tris-HCl pH 7.5 5 mMMgCl₂, 100 mM NaCl) containing 1250 U of BENZONASE® endonuclease (SigmaAldrich, St. Louis Mo.). The slurry was stirred for 1 hr at 37° C. andcentrifuged. The inclusion bodies were washed by resuspension indeionized water with a homogenizer and harvested by centrifugation for10 min at 10,000 rpm.

Example 8 Improved Acid Cleavage for the Production of Peptide HC353

The purpose of this experiment is to show that the new acid cleavagesequences identified in Example 4 with peptide TBP1 allow for improvedacid cleavage when another peptide is fused to the insolubility tag andtherefore are of general use. Improved acid cleavage means faster atequal pH and temperature, at a lower temperature for equal pH andincubation time or at a higher pH for equal temperature and incubationtime.

Each inclusion body paste was resuspended to 10% w/v in water containing50 mM NaCl. Each slurry (70 mL) was acidified by addition of HCl to pH6, pH 5, pH 4, pH 3, or pH 2. The acidified slurries were incubated at60° C., 70° C. or 80° C. with periodic manual agitation. Aliquots of theacidified slurries were collected at 15, 30, 60, 90, 120, 180 and 240min and placed on ice. Once all the aliquots were collected, the sampleswere neutralized by addition of 1 M NaOH.

The efficiency of the acid cleavage was assessed by SDS polyacrylamidegel electrophoresis and the gels were stained with Coomassie blue. Thebands corresponding to HC353 and KSI which have similar MW, could beresolved easily because of the abnormally slow gel mobility of HC353.The summary data is provided in Table 9 and is shown in FIGS. 9, 10, 11,and 12 where in each of the figures arrows are used to indicate the fulllength fusion construct (“F”), the peptide of interest HC353 (“H”) andthe inclusion body tag KSI(C4E) (“K”). The data confirms the resultsobtained with another peptide fusion in Example 4 and indicates that theimproved acid cleavage sites can be used broadly for the production ofdifferent peptides. Table 9. Data summary of the variant acid cleavagesites showing fusion proteins more labile to acid pH (faster kinetics,less acidic pH or lower temperature).

Fastest time Lowest temp. Highest pH for for complete for completecomplete Cleavage digest digest digest Strain Sequence @ pH 2, 80° C. @pH 2, 4 hrs @ 80° C.,  ID (SEQ ID NO: ) (min) (° C.) 4 hrs (pH) LD1474DP 240 80 2 LR2050 DPDPPDPP  60 60 4 (SEQ ID NO: 5) LR2321 DPPDPPDP  9070 3 (SEQ ID NO: 7) LR1755 DPDPDP 120 70 3 (SEQ ID NO: 2)

1. A method of preparing at least one peptide of interest (“POI”) from afusion peptide comprising at least one POI, comprising: a) providing arecombinant cell synthesizing the fusion peptide of claim 21 b)contacting the fusion peptide with a solution of sufficiently acidic pHso that linker L is cleaved, and c) isolating the at least one POI. 2.(canceled)
 3. The method of claim 1 wherein the recombinant cell is arecombinant microbial cell.
 4. The method of claim 3 wherein therecombinant microbial cell is a recombinant yeast cell.
 5. The method ofclaim 3 wherein the recombinant microbial cell is a recombinantbacterial cell.
 6. The method of claim 1 wherein the acid-cleavablelinker is cleaved by incubating the fusion peptides at a pH in the rangefrom about pH 1 to about pH
 4. 7. The method of claim 1 wherein theacid-cleavable linker is cleaved by incubating the fusion peptides at apH in the range from about pH 2 to about pH
 4. 8. The method of claim 1wherein the acid-cleavable linker is cleaved by incubating the fusionpeptides at a pH in the range from about pH 3 to about pH
 4. 9. Themethod of claim 1 wherein the acid-cleavable linker is cleaved byincubating the fusion peptides at a pH of about
 4. 10. The method ofclaim 1 wherein the acid-cleavable linker is cleaved by incubating thefusion peptides at a temperature of about 40° C. to about 90° C.
 11. Themethod of claim 1 wherein the acid-cleavable linker is cleaved byincubating the fusion peptides at a temperature of about 50° C. to about80° C.
 12. The method of claim 1 wherein the acid-cleavable linker iscleaved by incubating the fusion peptides at a temperature of about 60°C. to about 70° C.
 13. The method of claim 1 wherein the acid-cleavablelinker is cleaved by incubating the fusion peptides at a temperature ofabout 60° C.
 14. The method of claim 1 wherein the acid-cleavable linkeris cleaved by incubating the fusion peptides at a pH of about pH 2 toabout pH 4 using a temperature of about 50° C. to about 80° C.
 15. Themethod of claim 1, wherein PEP1 and PEP2 are both POIs.
 16. The methodof claim 15, wherein the fusion peptide is soluble in the recombinantcell.
 17. The method of claim 15, wherein the fusion peptide isinsoluble in the recombinant cell.
 18. The method of claim 17, whereincleaving the fusion peptide under acidic conditions renders the at leastone POI soluble.
 19. The method of claim 1, wherein either PEP1 or PEP2of the fusion peptide comprises an inclusion body tag, therebycomprising a non-POI portion of the fusion peptide.
 20. The method ofclaim 19, wherein the non-POI portion remains insoluble after cleavingthe fusion peptide.
 21. A fusion peptide comprising two peptidesseparated by an acid-cleavable linker according to the following generalformula:PEP1-L-PEP2 wherein, a) PEP1 and PEP2 are independently functionalpeptides wherein at least one is a peptide of interest (“POI”); and b) Lis an acid-cleavable linker comprising a peptide selected from the groupconsisting of: (SEQ ID NO: 1) A. DPDP, (SEQ ID NO: 2) B. DPDPDP, and(SEQ ID NO: 3) C. DPDPDPDP,

wherein D is aspartic acid and P is proline.
 22. (canceled)
 23. Thefusion peptide of claim 21 wherein PEP1 and PEP2 are nonidentical. 24.The fusion peptide of claim 23, wherein the fusion peptide is soluble ina recombinant cell.
 25. The fusion peptide of claim 24 wherein therecombinant cell is a recombinant microbial cell.
 26. The fusion peptideof claim 25 wherein the recombinant microbial cell is a recombinantbacterial cell.
 27. The fusion peptide of claim 25 wherein therecombinant microbial cell is a recombinant yeast cell.
 28. The fusionpeptide of claim 23, wherein the fusion peptide is insoluble in arecombinant cell.
 29. The fusion peptide of claim 28 wherein therecombinant cell is a recombinant microbial cell.
 30. The fusion peptideof claim 29 wherein the recombinant microbial cell is a recombinantyeast cell.
 31. The fusion peptide of claim 30 wherein the recombinantmicrobial cell is a recombinant bacterial cell.
 32. The fusion peptideof claim 23 wherein either of PEP1 or PEP2 comprises an inclusion bodytag (“IBT”).
 33. The fusion peptide of claim 32 wherein the fusionpeptide is present in inclusion bodies.
 34. The fusion peptide of claim23 wherein the acid-cleavable linker is cleaved by incubation at a pH inthe range from about pH 1 to about pH
 4. 35. The fusion peptide of claim23 wherein the acid-cleavable linker is cleaved by incubating at atemperature of about 40° C. to about 90° C.
 36. The fusion peptide ofclaim 35 wherein the acid-cleavable linker is cleaved by incubating at atemperature of about 50° C. to about 80° C.
 37. The fusion peptide ofclaim 36 wherein the acid-cleavable linker is cleaved by incubating at atemperature of about 60° C. to about 70° C.
 38. The fusion peptide ofclaim 23 wherein the acid-cleavable linker is cleaved by incubating at apH of about pH 2 to about pH 4 and at a temperature of about 50° C. toabout 80° C.
 39. A recombinant cell expressing a fusion protein havingthe structurePEP1-L-PEP2 wherein, i) PEP1 and PEP2 are independently functionalpeptides, one of which is a POI; and ii) L is an acid-cleavable linkercomprising a peptide selected from the group consisting of:(SEQ ID NO: 1) A. DPDP, (SEQ ID NO: 2) B. DPDPDP, and (SEQ ID NO: 3)C. DPDPDPDP,

wherein D is aspartic acid and P is proline; and wherein the expressedfusion peptide is present in the recombinant cell.
 40. (canceled) 41.The recombinant cell of claim 39 wherein the recombinant cell is arecombinant microbial cell.
 42. The recombinant cell of claim 41 whereinthe recombinant cell is a recombinant bacterial cell.
 43. Therecombinant cell of claim 41 wherein the recombinant cell is arecombinant microbial cell is a recombinant yeast cell.
 44. Anacid-cleavable peptide linker comprising a peptide selected from thegroup consisting of: (SEQ ID NO: 1) A. DPDP, (SEQ ID NO: 2) B. DPDPDP,and (SEQ ID NO: 3) C. DPDPDPDP,

wherein D is aspartic acid and P is proline. 45-46. (canceled)